Glycosylation Methods and Analysis Sigma

Glycoprotein
Analysis
1st Edition
M A N U A L
Authors:
Robert Gates
Elner Rathbone
Lisa Masterson
Ian Wright
Asgar Electricwala
Glycan
Classification
Glycoprotein
Purification
Deglycosylation
Strategies
Detection
Glycan Analysis
Product Directory
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Glycoprotein Analysis
Application Note
PNGase F Enzyme:
Efficient N-Deglycosylation
in Glycoprotein Analysis
By Elner Rathbone, Asgar Electricwala, and Ian Wright
Sigma-Aldrich Corporation, Poole, England
Introduction
Glycosylation is one of the most common post-translational
modifications of proteins in eukaryotic cells. These glycoproteins are involved in a wide range of biological functions such as receptor binding, cell signaling, immune
recognition, inflammation, and pathogenicity. Mammalian
glycoproteins contain three major types of oligosaccharides (glycans): N-linked, O-linked, and glycosylphosphatidylinositol (GPI) lipid anchors. N-Linked glycans are attached
to the protein backbone via an amide bond to an asparagine
residue in an Asn-Xaa-Ser/Thr motif, where X can be any
amino acid, except Pro. O-Linked glycans are linked via the
hydroxyl group of serine or threonine. Variation in the
degrees of saturation at available glycosylation sites results
in heterogeneity in the mass and charge of glycoproteins.
To study the structure and function of a glycoprotein, it is
often desirable to remove all or just a select class of glycans.
This approach allows the assignment of specific biological
functions to particular components of the glycoprotein.
The removal of N-linked glycans from glycoproteins eliminates heterogeneity in matrix-assisted laser desorption/
ionization-time of flight (MALDI-TOF) MS analysis. Also,
removal of glycans may enhance or reduce the blood
clearance rate and/or potency of a glycoprotein therapeutic. Although sites of potential N-glycosylation can be
predicted from the consensus sequence Asn-Xaa-Ser/Thr,
it cannot be assumed that a site will actually be glycosylated. Therefore the sites of glycan attachment must be
identified and characterized by analytical procedures.
Peptide-N-glycosidase F (PNGase F) is one of the most widely
used enzymes for the deglycosylation of glycoproteins.
The enzyme releases asparagine-linked (N-linked) oligosaccharides from glycoproteins and glycopeptides. A tripeptide
with the glycan-linked asparagine as the central residue
is the minimum substrate for PNGase F. The glycan can
be a high-mannose, hybrid, or complex type. However,
N-glycans with fucose linked a(1,3) to the Asn-bound
N-acetylglucosamine are resistant to the action of PNGase F.
MALDI-TOF MS is a widely used technique for rapid identification of proteins separated by gel electrophoresis.
Glycopeptides, however, suffer from signal suppression
during MS analysis due to the heterogeneity of the

attached glycans. In this application note, we have used
PNGase F enzyme (Product Code P 7367) for the deglycosylation of a model glycoprotein, a1-antitrypsin, and have
identified and localized N-glycosylation sites by MALDI-MS
peptide mapping. The GlycoProfile I Enzymatic In-Gel
N-Deglycosylation Kit (Product Code PP 0200) conveniently
provides quality-tested reagents and a detailed protocol
for in-gel protein deglycosylation.
Efficient and convenient in-gel protein deglycosylation
A specific example demonstrating the use of the enzyme
is the deglycosylation of the glycoprotein a1-antitrypsin.
After reduction and alkylation (using tributylphosphine and
iodoacetamide contained in the ProteoPrep Reduction
and Alkylation Kit, Product Code PROT-RA), samples are
mixed with SDS-PAGE sample buffer and separated on
10% BisTris NuPAGE gel (Invitrogen Corp., Carlsbad, CA)
using MOPS running buffer. The gel is stained with 0.1%
Coomassie blue stain and destained.
The glycoprotein band from the 1D gel (or a spot from
a 2D gel) is carefully cut out and sliced into sections. The
pieces are destained, dried, and incubated with PNGase F.
The liquid surrounding the gel pieces is removed and
either discarded or retained for gylcan analysis if required.
The dried gel pieces are incubated with trypsin (Product
Code T 6567) and the surrounding liquid containing the
tryptic peptides is removed for further analysis.
Facile sample preparation for mass spectrometry
Sample preparation is simplified since the enzyme formulation does not contain detergents or stabilizers that
could interfere with analysis by MS. The solution of tryptic
peptides is concentrated and desalted on a C18 ZipTip
(Millipore Corporation, Billerica, MA) prior to analysis.
MS analysis was carried out on a MALDI-TOF instrument
fitted with a 337-nm UV laser. Peptides were analyzed
in positive ion reflectron mode using a-cyano-4-hydroxycinnaminic acid as the matrix. Spectra were acquired
over a mass range of 4000 m/z with matrix suppression
set at 800 mass units. Data analysis was carried out using
ProteinLynx software (Waters Corporation, Milford, MA).
Summary
PNGase F is an effective enzyme for the release of N-linked
glycans from glycoproteins, in gel or in solution. The formulation employed for PNGase F (Product Code P 7367)
provides a convenient, stable, freeze-dried product that
is compatible with MS analysis. This enzyme is also included
in the GlycoProfile In-Gel and GlycoProfile In-Solution
Deglycosylation Kits, which are composed of Proteomics
grade, high purity components for N-deglycosylation and
tryptic digestion.
Table of Contents
Overview
Key to Monosaccharide Symbols,
Abbreviations and Projections . . . . . . . . . . . . 02
Classification and Structure of
Glycan Components
N-Linked Glycans . . . . . . . . . . . . . . . . . . . 05
O-Linked Glycans . . . . . . . . . . . . . . . . . . . 09
GPI Anchored Proteins . . . . . . . . . . . . . . . 12
Glycoprotein Purification
m-Aminophenylboronate Matrices . . . . . . . . 13
Lectin Matrices . . . . . . . . . . . . . . . . . . . . . . . . 13
Glycoprotein Detection
Colorimetric Detection on PAGE and Blots .
Fluorescent Detection on PAGE . . . . . . . . .
Biotin Labeling on Blots . . . . . . . . . . . . . . .
Glycoprotein Standards . . . . . . . . . . . . . . .
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Chemical Deglycosylation Strategies

Hydrazinolysis. . . . . . . . . . . . . . . . . . . . . . . . . 22
Alkaline b-Elimination . . . . . . . . . . . . . . . . . . 23
Trifluoromethanesulfonic Acid . . . . . . . . . . . . 23
Enzymatic Deglycosylation Strategies
N-Linked Glycan Enzymatic Hydrolysis .
Native and Sequential Hydrolysis . . . . .
O-Linked Glycan Enzymatic Hydrolysis .
Endoglycosidases . . . . . . . . . . . . . . . . .
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Glycan Labeling and Analysis

Glycan Labeling . . . . . . . . . . .
HPLC Analysis . . . . . . . . . . . . .
Mass Spectrometry . . . . . . . . .
NMR Analysis . . . . . . . . . . . . .
Electrophoresis . . . . . . . . . . .
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Glycan and Carbohydrate Standards

N-Linked High Mannose. . . . . . . . . . .
N-Linked Hybrid . . . . . . . . . . . . . . . . .
N-Linked Complex . . . . . . . . . . . . . . .
N-Linked Fragments . . . . . . . . . . . . . .
O-Linked Neutral Glycans . . . . . . . . . .
O-Linked Sialylated Glycans . . . . . . . .
Blood Group Antigens . . . . . . . . . . . .
Lewis and Cell Adhesion Glycans . . . .
Gal a(1,3) Gal Antigens . . . . . . . . . . .
Monosaccharides . . . . . . . . . . . . . . . .
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Glycobiology Product Directory

Additional Information Sources:
Enzyme Explorer. . . . . . . . . . . . . . . . . .
Complete List of Glycolytic Enzymes . .
GPI Anchor Enzymes. . . . . . . . . . . . . . .
Glycosyltransferases . . . . . . . . . . . . . . .
Inhibitors of Carbohydrate Metabolism .
Glycolytic Enzyme Substrates . . . . . . . .
Neoglycoproteins . . . . . . . . . . . . . . . . .
Lectins. . . . . . . . . . . . . . . . . . . . . . . . . .
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Overview
One of the distinguishing features of the
proteome in eukaryotic cells is that most
proteins are subject to post-translational
modification, of which glycosylation is the
most common form. It is estimated that
more than half of all proteins that have
been characterized are glycoproteins. The
carbohydrate components of glycoproteins
perform critical biological functions in
protein sorting, immune and receptor
recognition, inflammation, pathogenicity,
metastasis, and other cellular processes.
Mammalian glycoproteins contain three
major types of oligosaccharides (glycans):
N-linked, O-linked, and glycosylphosphatidylinositol (GPI) lipid anchors. N-Linked glycans
are linked to the protein backbone via an
amide bond to asparagine residues in an
Asn-X-Ser/Thr motif, where X can be any
amino acid, except Pro. O-Linked glycans
are linked to the hydroxyl group of serine
or threonine. GPI-anchored proteins are

attached at their carboxy-terminus through a
phosphodiester linkage of phosphoethanolamine to a trimannosyl glucosamine core
structure. The reducing end of the latter
moiety is bound to the hydrophobic region
of the membrane via a phosphatidylinositol group.
Variations in structure and degree of glycosylation site saturation can contribute to
overall mass heterogeneity. The terminal
residues of these glycans are commonly
N-acetylneuraminic acid (sialic acids). The
degree of sialylation affects both the mass
and charge of a glycoprotein. Other modifications to the protein such as sulfation
or phosphorylation also affect charge.
O-Linked glycans often have lower mass
than N-linked structures, but can be more
abundant and heterogeneous.
Key to Monosaccharide Symbols, Abbreviations, and Projections

Symbols, Structure Projections, and Abbreviations for Monosaccharides
Monosaccharide
3-D Chair projection
Haworth projection
OH
HO
CH2OH
O
OH
HO
OH
HO
OH
Fischer projection
H
HO
OH
OH
OH
Glc
OH
CH2OH
b-D-Glucose (Glc)
OH
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OH
CH2OH
HO
HO
HO
b-D-Mannose (Man)
Symbol
OH
OH
OH
OH
HO
HO
HO
OH
OH
H
CH2OH
Man

Monosaccharide
Haworth projection
HO
OH
CH2OH
HO
OH
Symbol
H
O
OH
OH
OH
OH
HO
Fischer projection
HO
HO
Gal
OH
H
OH
CH2OH
b-D-Galactose (Gal)
OH
CH2OH
O
HO
OH
HO
HO
OH
NHAc
OH
NHAc
HO
OH
H
NHAc
GlcNAc
OH
H
CH2OH
b-D-N-Acetylglucosamine (GlcNAc)
CH2OH
OH
HO
OH
OH
HO
H
OH
HO
OH
NHAc
NHAc
HO
HO
GalNAc
NHAc
b-D-N-Acetylgalactosamine
(GalNAc)
CH2OH
HO
HO
OH
OH
OH
b-D-Xylose (Xyl)
H
HO
OH
OH
OH
OH
OH
Xyl
H
H
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O
HO

Monosaccharide
Haworth projection
Fischer projection
CO2H
HO
CH2OH
OH
HO
CO2H
AcHN HO
AcHN
OH
OH
OH
CO2H
OH
OH
HO
OH
a-N-Acetylneuraminic acid
Sialic Acid (NeuNAc)
OH
OH
CH2OH
CH2OH
CO2H
HO
O
OH
OH
O
HO
OH
OH
HO
HO
OH
OH
H
OH
H
CO2H
HO
O
OH
HO2C
OH
HO
OH
OH
IdoA
OH
OH
H
CO2H
OH
H3C
HO
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OH
a-L-Iduronic acid (IdoA)
a-L-Fucose (Fuc)
CH2OH
OH
HO
HO
GlcA
OH
b-D-Glucuronic acid (GlcA)
HO
NeuNAc
AcHN
OH
Symbol
CH3
O
OH
OH
HO
HO
OH
OH
OH
OH
OH
H
CH3
Fuc
Classification and Structure of Glycan Components

N-Linked Glycans
All eukaryotic cells express N-linked glycoproteins. Protein
glycosylation of N-linked glycans is actually a co-translational
event, occurring during protein synthesis. N-linked glycosylation requires the consensus sequence Asn-X-Ser/Thr.
Glycosylation occurs most often when this consensus
sequence occurs in a loop in the peptide. Oligosaccharide
intermediates destined for protein incorporation are synthesized by a series of transferases on the cytoplasmic
side of the endoplasmic recticulum (ER) while linked to
the dolichol lipid. Following the addition of a specific
number of mannose and glucose molecules, the orientation of the dolichol precursor and its attached glycan shifts
to the lumen of the ER where further enzymatic modification occurs. The completed oligosaccharide is then
transferred from the dolichol precursor to the Asn of the
target glycoprotein by oligosaccharyltransferase (OST).
Further processing includes trimming of residues such as
glucose and mannose, and addition of new residues via
transferases in the ER and, to a great extent, in the Golgi.
In the Golgi, high mannose N-glycans can be converted
to a variety of complex and hydrid forms which are unique
to vertibrates.
Inhibition or elimination of glycosylation in the study of

N-linked glycans can be brought about by a number of
compounds. In the presence of compactin, coenzyme Q,
and exogenous cholesterol, N-glycosylation is greatly
inhibited. Treatment with tunicamycin completely blocks
deglycosylation in that it inhibits GlcNAc C-1-phosphotransferase, which is critical in the formation of the dolichol
precursor necessary for synthesizing of N-glycans.
The diverse assortment of N-linked glycans are based on
the common core pentasaccharide, Man3GlcNAc2. Further
processing in the Golgi results in three main classes of
N-linked glycan sub-types; High-mannose, Hybrid, and
Complex. Complex glycans contain the common trimannosyl core. Additional monosaccharides may occur in
repeating lactosamine units. Additional modifications may
include a bisecting GlcNAc at the mannosyl core and/or
a fucosyl residue on the innermost GlcNAc. Complex
glycans exist in bi-, tri- and tetraantennary forms.
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Basic N-linked Structure
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High-Mannose Structure
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Hybrid Structure
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Complex Structure (tetraantennary)
O-Linked Glycans
O-Linked glycans are usually attached to the peptide chain
through serine or threonine residues. O-Linked glycosylation is a true post-translational event and does not require
a consensus sequence and no oligosaccharide precursor
is required for protein transfer. The most common type
of O-linked glycans contain an initial GalNAc residue (or
Tn epitope), these are commonly referred to as mucin-type
glycans. Other O-linked glycans include glucosamine, xylose,
galactose, fucose, or manose as the initial sugar bound to
the Ser/Thr residues. O-Linked glycoproteins are usually
large proteins (>200 kDa) that are commonly bianttennary
with comparatively less branching than N-glycans.
Glycosylation generally occurs in high-density clusters
and may contribute as much as 50-80% to the overall mass.
O-Linked glycans tend to be very heterogeneous, hence
they are generally classified by their core structure. Nonelongated O-GlcNAc groups have been recently shown to
be related to phosphorylation states and dynamic processing
related to cell signaling events in the cell. O-Linked glycans

are prevalent in most secretory cells and tissues. They
are present in high concentrations in the zona pelucida
surrounding mammalian eggs and may funtion as sperm
receptors (ZP3 glycoprotein). O-Linked glycans are also
involved in hematopoiesis, inflammation response mechanisms, and the formation of ABO blood antigens.
Elongation and termination of O-linked glycans is carried
out by several glycosyltransferases. One notable core
structure is the Gal-b(1-3)GalNAc (core 1) sequence that
has antigenic properties. Termination of O-linked glycans
usually includes Gal, GlcNAc, GalNAc, Fuc, or sialic acid.
By far the most common modification of the core
Gal-b(1-3)-GalNAc is mono-, di-, or trisialylation. A less
common, but widely distributed O-linked hexasaccharide
structure contains b(1-4)-linked Gal and b(1-6)-linked
GlcNAc as well as sialic acid.
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Di- and Trisialated O-Linked Core
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O-Linked Core 2 Hexasaccharide
Core 1
Core 2
Core 3
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Core 4
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Core 5
Core 6
Core 7
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Core 8
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GPI Anchor Glycoproteins

Glycosylphosphatidylinisotol (GPI) anchored proteins are
membrane bound proteins found throughout the animal
kingdom. GPI anchored proteins are linked at their carboxyterminus through a phosphodiester linkage of phosphoethanolamine to a trimannosyl-non-acetylated glucosamine
(Man3-GlcN) core. The reducing end of GlcN is linked to
phosphatidylinositol (PI). PI is then anchored through
another phosphodiester linkage to the cell membrane
through its hydrophobic region. Intermediate forms are
also present in high concentrations in microsomal preparations. The Man3-GlcN oligosaccharide core may undergo
various modifications during secretion from the cell.
Their functionality ranges from enzymatic to antigenic

and adhesion. They contribute to the overall organization
of membrane bound proteins and are important in apical
protein postioning. GPI-anchored proteins also play a
critical role in a variety of receptor-mediated signal
transduction pathways.
Release of GPI anchored proteins can be accomplished by
treatment with Phospholipase C, Phosphatidylinositolspecific (PLC-PI) (Product Codes P 5542 and P 8804). The
enzyme specifically hydrolyzes the phosphodiester bond
of phosphatidylinositol to form a free 1,2-diacylglycerol
and glycopeptide-bound inositol cyclic-1,2-phosphate.
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GPI Anchor
13
Purification of proteins selectively utilizing their glycan

component as a capture target is commonly done utilizing affinity chromatography. The most popular affinity
matrices are m-aminophenylboronic acid agarose and
the immobilized lectins, Con A and Wheat Germ.
m-Aminophenylboronic Acid Matrices

m-Aminophenylboronic acid matrices are capable of
forming temporary bonds with any molecule that
contains a 1,2-cis-diol group.
Procedure
Equilibration buffers should be of low ionic strength,
with pH 7-9.
For a column volume of 1 ml, apply 1-2 mg of protein in
approximately 250 ml of buffer: 50 mM taurine/NaOH,
pH 8.7, containing 20 mM MgCl2.
Optimize the column flow rate to 2 ml/hour, collecting
2 ml fractions.
Elute the bound protein using the same buffer with
50 mM sorbitol or 50 mM Tris/HCl added.
References
1. Mallia, A.K. et al., Anal. Letters, 14 (B8), 649-661 (1981).
2. Immobilized Affinity Ligand Techniques, Hermanson, G.T., et al.,
Eds. (Academic Press, 1992), pp. 338, 339-392.
3. Affinity Chromatography: A Practical Approach, P.D.G Dean, W.S.
Johnson and F. A. Middle, Eds., (IRL Press, 1985), p. 133.
Aminophenylboronate Matrices
A 8530
m-Aminophenylboronic Acid
Affinity Medium
Matrix: cross-linked 6% beaded agarose

Activation: epoxy, with attachment through the amino group, with a 12-atom spacer
Ligand immobilized: 5-20 mmoles per ml
Form: (light pink) suspension in 0.5 M NaCl, with 0.1 M sodium acetate, pH 5.0
Synonym: PBA-agarose
A 4046
Affinity Medium
Matrix: acrylic beads

Activation: oxirane, with attachment through the amino group, with a 5-atom spacer
Ligand immobilized: 300-600 mmoles per gram
Form: lyophilized powder
Swelling: 1 g swells to approximately 4 ml
A 8312
Affinity Medium
Matrix: 6% beaded agarose

Activation: epichlorohydrin, with attachment through amino group with a 9-atom spacer
Ligand immobilized: 40-90 mmoles per ml
Binding Capacity: 8-14 mg Peroxidase Type VI per ml
Form: suspension in water containing 0.002% chlorhexidine diacetate.
(For additional Lectins see page 80)

Concanavalin A matrices bind specifically to mannosyl
and glucosyl residues of polysaccharides and glycoproteins. Unmodified hydroxyl groups at the C3, C4, and
C6 positions of D-glucopyranosyl or D-mannopyranosyl
rings may be essential for binding. Con A matrices have
been used with SDS (0.05%) and TRITON X-100.
Procedure
Equilibration and Binding:
Pre-wash column with 5 column volumes of wash
solution (1 M NaCl, 5 mM MgCl2, 5 mM MnCl2, and
5 mM CaCl2).
Equilibrate column in the buffer of choice (pH ranges
generally between 6.5 to 7.5 although buffers as low as
pH 4.1 and as high as 9.0 have been used successfully).
A commonly used starting buffer is 20 mM Tris, pH 7.4,
containing 0.5 M NaCl.
Load sample solution in equilibration buffer (protein
concentrations 1-20 mg/ml, free of particulates).
Wash the resin with equilibration buffer until eluent is
protein free.
Elution:
Elute the target protein with gradient or step-wise
elution with methyl a-D-glucopyranoside or methyl
a-D-mannopyranoside, glucose, or mannose (5 mM 500 mM).
Maximum recovery and cleaning of the resin may be
achieved by using 1 M sucrose, glucose, mannose, or
corresponding a-methyl glycoside. The addition of
chaotropic agents (0.5 M to 6 M) may also be required
for maximum recovery, but these denaturing conditions
may severely damage the resin. Therefore they should
only be used as a last resort.
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Lectin Matrices
14
Con A Matrices
C 9017
Concanavalin A Immobilized
Matrix: Sepharose 4B
Activation: cyanogen bromide
Ligand immobilized: 10-15 mg per ml
Binding Capacity: 20-45 mg thyroglobulin per ml
Form: suspension in 0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM each of
CaCl2, MgCl2, and MnCl2 and 0.01% thimerosal
C 6170
Concanavalin A Immobilized
Matrix: 4% beaded agarose

Ligand immobilized: approx. 15 mg per ml
Binding Capacity: 6 mg yeast mannan per ml
Form: suspension in 0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM each of
CaCl2, MgCl2, and MnCl2 and 0.02% thimerosal
References
1. Yahara, I. and Edelman, G.M., Restriction of the mobility of lymphocyte immunoglobulin receptors by concanavalin A. Proc. Nat.
Acad. Sci. USA, 69(3), 608-612 (1972).
2. Li, Y., et al., The p185neu-containing Glycoprotein Complex of a
Microfilament-associated Signal Transduction Particle. J. Biol.
Chem., 274(36), 25651-25658 (1999).
3. Romero, P.A., et al., Glycoprotein biosynthesis in Saccharomyces

cerevisiae. Partial purification of the alpha-1,6-mannosyltransferase that initiates outer chain synthesis. Glycobiology, 4(2),
135-140 (1994).
Wheat Germ (Triticum vulgaris) matrices are specific for

GlcNAc2 or NeuNAc residues.
Elution:
Elute the target protein with gradient or step-wise
elution with equilibration buffer containing 100 mg/ml
N-acetylglucosamine (Product Code A 8625).
Procedure
Equilibration and Binding:
Wash and equillibrate column with 5 column volumes
of wash solution (0.05 M sodium phosphate, pH 7.0,
containing 0.2 M NaCl).
Load sample solution in equilibration buffer (protein
concentrations 1-20 mg/ml, free of particulates).
Wash the resin with equilibration buffer until eluent is
protein free.
WGA Matrices
L 1882
Wheat Germ (Triticum vulgaris)

Lectin Immobilized
Matrix: Cross-Linked 4% beaded agarose

Ligand immobilized: 5-10 mg per ml
Form: Suspension in 1.0 M NaCl and 0.02% thimerosal
L 1394
Wheat Germ (Triticum vulgaris)

Lectin Immobilized
Matrix: 6% agarose macrobeads

Ligand immobilized: Approx. 6 mg per ml
Binding Capacity: 1-2 mg ovomucoid per ml
Form: Suspension in 0.9% NaCl and 0.01% thimerosal
References
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1. Lotan, R., et al., Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin
affinity chromatography for the purification of membrane glycoproteins. Biochem., 16, 1787-1794 (1977).
2. Janicott, M., et al., The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and
IGF-2 in Xenopus laevis oocytes. J. Biol. Chem., 266, 9382 (1991).
15
Initial detection of glycoproteins in vitro is routinely accomplished on SDS-PAGE gels and

Western blots. Cellular localization of glycoproteins is normally accomplished utilizing
lectin fluorescent conjugates.
For a complete list of lectins including fluorescent labeled lectins, see page 80.
Glycoprotein Detection Kit

Product Code GLYCO-PRO
Product Description
The Glycoprotein Detection Kit provides a system to easily
detect the sugar moieties of glycoproteins on SDS-PAGE or
on Western blotting membranes. This detection system is
a modification of Periodic acid-Schiff (PAS) methods and
yields magenta bands with a light pink or colorless background. The detection limits have been found to be
25-100 ng of carbohydrates depending on the nature
and the degree of glycosylation of proteins. Peroxidase
from horseradish, reported as having a carbohydrate

content of approximately 16%, is used as a positive
control in the kit. The table below describes the steps
and time required when utilizing this kit.
Contains sufficient materials to stain 10 mini gels
(8 x 10 cm) or 5 large gels (16 x 18 cm) or same sizes
of blotting membranes.
Components
Oxidation Component (Periodic Acid)
Reduction Component (Sodium Metabisulfite)
Schiffs Reagent, Fuchsin-Sulfite Reagent
Peroxidase
Steps
Time for gel thickness 0.5-0.75 mm or for membrane
Time for gel thickness 1.0-1.5 mm
1. Fixing
30 min.
60 min.
2. Washing
2 x 10 min.
2 x 20 min.
3. Oxidation
30 min.
60 min.
4. Washing
2 x 10 min.
2 x 20 min.
5. Staining
1-2 hours or until bands turn magenta
1-2 hours or until bands turn magenta
6. Reduction
60 min.
120 min.
7. Washing
Band color will intensify with changes of fresh water
Band color will intensify with changes of

fresh water
8. Storage
overnight
overnight
sigma-aldrich.com
Colorimetric Detection on PAGE and

Western blots
16
Fluorescent Detection on PAGE Gels
GlycoProfile III
Fluorescent Glycoprotein Detection Kit
8 Product Code PP0300
Product Description
GlycoProfile III is designed for the fluorescent in-gel
detection of glycosylated proteins utilizing standard
UV-transillumination. Following SDS-PAGE, proteins are
fixed in the gel with an acetic acid/methanol solution.
The carbohydrates on the proteins are oxidized to aldehydes with periodic acid. A hydrazide dye is reacted with
the aldehydes, forming a stable fluorescent conjugate.
This allows for the specific, sensitive detection of the
glycoproteins directly in gels. This kit can also be used
to detect glycoproteins after Western transfer to PVDF
membranes.
Ovalbumin (glycosylated and phosphorylated)

b-Casein (not glycosylated, phosphorylated)
RNase B (glycosylated, not phosphorylated)
Rabbit
Mouse
Rabbit
Figure 1. PTM Marker (2 ml of a 6-fold dilution), containing glycosylated and non-glycosylated proteins, was separated by electrophoresis
on a 420% SDS-PAGE gel. The gel was stained for glycoproteins
with GlycoProfile III (left), imaged, and then stained for total protein
with EZBlue Gel Staining Reagent (right). The glycoproteins appear
as bright fluorescent bands. Each band represents approximately
300 ng of protein.
Mouse
The classical method for in-gel carbohydrate detection

uses Periodic Acid-Schiff reagent (PAS). It has a detection
limit of 25-100 ng of carbohydrate. PAS staining of glycoproteins is very selective, but lacks the sensitivity of fluorescent detection (5-25 ng of carbohydrate). The limit of
detection varies with the glycoprotein and the degree
and type of glycosylation. Typical detection limits observed
with the ProteoProfile PTM Marker (P 1745) are 150 ng
of ovalbumin (5 ng of carbohydrate) and 150 ng of RNase B
(30 ng carbohydrate). The detection limits are 5-10 times
lower than those observed with the Periodic Acid-Schiff
reagent.
BSA (not glycosylated, not phosphorylated)
Sufficient reagents are supplied for 10 mini gels.

Components
IgG Heavy Chain (glycosylated)
Oxidation Reagent
Glycoprotein Staining Reagent
Staining Buffer
IgG Light Chain
ProteoProfile PTM Marker

Designed as both a positive and negative control for SDS-PAGE gels
and Western blots of proteins with post-translation modifications.
sigma-aldrich.com
Specificity
Although this staining procedure is quite selective for
glycoproteins, some non-specific protein staining may
occur and may be more pronounced in some gel formulations. Staining the gel with EZBlue Gel Staining
Reagent after fluorescent imaging will allow identification of non-specifically stained proteins.
An alternative method is to run duplicate gels and fluorescently stain the second gel omitting the oxidation
step. Any fluorescent staining will be non-specific.
Figure 2. Mouse IgG and rabbit IgG were separated on a 420%

SDS-PAGE gel and stained in the same manner as the gel in Figure 1.
The IgG heavy chains, which are glycosylated, react strongly with
the fluorescent detection reagent. 2.5 mg of protein was applied
to each lane.
17
colorimetric TMB or chemiluminescent CPS-1 peroxidase
substrate solutions. Alkaline phosphatase conjugated
streptavidin can also be used to probe the blots. Typical
detection limits for glycoproteins using this is method
are approximately 50-100 ng.
Biotin-hydrazide modification of periodate oxidized glycans can be utilized to label glycoproteins on Western
blots. The blots can then be probed with streptavidinperoxidase.1 Detection can be accomplished with either
Glycan
Optimal results are obtained on PDF membranes.
Biotin Labeled Glycan
Periodate
Oxidized
Glycan
O
Periodate
Detection of Biotin Labeled

Glycoproteins on Western blots
Biotin Hydrazide
CH
CH
NH
NH
OH
OH
HN
NH
O=C
C=O
H2C
CH2 = CH2 = CH2 = CH2 = C = N = NH2

H
H2C
CH2
CH2
H2C
H2C
CH2
H
N
S
N
H
CH2
H
N
N
H
Detection Products
B 7639
Biotin Hydrazide
S 5512
Streptavidin, Peroxidase Labeled
S 1878
Sodium (meta) Periodate
T 0565
Tetramethylbenzidine Liquid Substrate System
CPS1-60
Chemiluminescent Peroxidase Substrate 60 ml
CPS1-120
CPS1-300
Reference
sigma-aldrich.com
1. Bayer, E.A., et al., Meth. Enzymol., 184, 415 (1990).
18
Glycoproteins and
Glycoprotein Standards
There are many methods currently utilized in glycoprotein
detection and identification. Among the most common
are SDS-PAGE, Mass Spectroscopy, HPLC, NMR, and
Western blot. It is helpful and often necessary to include
glycoproteins as standards and/or molecular weight markers.
In biological assays involving cell or animal models, the

physiologic activity of glycoproteins is of primary interest.
Sigma-Aldrich offers a broad variety of glycoproteins for
analytical and biological use as standards, controls, and
bioactive components. The following products are an
abbreviated list of the most popular glycoproteins in
common analytical and biological assays. For additional
products please visit our website at sigma-aldrich.com.
Preferred Glycoprotein Standards

I 0408
8
Invertase Glycoprotein Standard
Invertase is an enzyme that catalyzes the hydrolysis of sucrose into fructose and glucose.
Invertase Glycoprotein Standard is the periplasmic (glycosylated form, external invertase)
with 50% of its mass as polymannan. The periplasmic invertase molecule can exist in a
number of association states each a multiple of the core glycosylated monomer, a 60 kDa
peptide plus oligosaccharide chains and, depending on extraction, purification, and storage
conditions, will exist as a dimer, tetramer, hexamer, or octamer. Since yeast can provide an
alternative system for protein glycosylation that is similar to mammalian systems, periplasmic invertase is often used as a model for the study of the function of oligosaccharides in
glycoproteins and for studies on glycoprotein biosynthesis.
R 1153
8
RNase B Glycoprotein Standard
Bovine pancreatic Ribonuclease B (RNase B) is a glycoprotein that contains only N-linked

glycans. It is a globular protein composed of a single domain that occurs naturally as a
lesser component in a mixture along with Ribonuclease A (RNase A) which is the nonglycosylated core form. RNase B contains a single glycosylation site at Asn34 at which
from five to nine mannose residues are attached to the chitobiose core, i.e. Man5GlcNAc2,
Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2 and Man9GlcNAc2. Due to the heterogeneity
in the glycosylation at Asn34, RNase B exists as five glycosylated variants, with a molecular
weight of approximately 15,000 Da.
sigma-aldrich.com
Key Glycoproteins for Scientific Research

G 9885
a1-Acid Glycoprotein, human
99%, Purified from Cohn Fraction VI
A 5566
Monoclonal Anti-a1-Acid
Glycoprotein antibody
produced in mouse, Clone AGP-47, Ascites fluid, liquid
A 0534
Anti-a1-Acid Glycoprotein antibody
produced in rabbit, IgG fraction of antiserum, Lyophilized powder
A 3418
Anti-a1-Acid Glycoprotein antibody
produced in goat, Whole antiserum, liquid
A 9285
a1-Antichymotrypsin
from human plasma
approx. 95% (SDS-PAGE) Lyophilized powder

Glycoprotein is specific inhibitor of chymotrypsin-like serine proteases.
Contains Tris buffer salt and NaCl
A 9024
a1-Antitrypsin
from human plasma
Salt-free, lyophilized powder, a1-Proteinase inhibitor, serine protease inhibitor; inhibits trypsin,
chymotrypsin, pancreatic and granulocytic elastase, and acrosin. Effective concentration
equimolar with proteinase. Chromatographically prepared and partially purified.
G 0516
a2-hs-Glycoprotein
from human plasma
minimum 90% (SDS-PAGE) Lyophilized from 20 mM Tris-HCl, pH 8.0, with 200 mM NaCl
A 1960
Aggrecan
from bovine articular cartilage
Lyophilized powder, salt essentially free

sterile-filtered (dialyzed against water)
A 2512
Albumin
from chicken egg white
(Ovalbumin)
Grade VI approx. 99% (agarose gel electrophoresis) Lyophilized powder

Extent of labeling 5-6 mol mannose per mol ovalbumin protein
A 5503
Albumin
(Ovalbumin)
Grade V minimum 98% (agarose gel electrophoresis) Lyophilized powder

salt essentially free
A 5378
Albumin
(Ovalbumin)
Grade III minimum 90% (agarose gel electrophoresis) Lyophilized powder
A 4781
Asialofetuin
from fetal calf serum
Type I, salt essentially free

N-acetylneuraminic acid <0.5%
19
Asialoglycophorin
from human blood Type MN
Lyophilized powder, Predominantly Asialoglycophorin A
G 7900
Monoclonal Anti-Glycophorin A
(a) antibody
Produced in mouse, Ascites fluid, Clone E4, Liquid
G 7650
Monoclonal Anti-Glycophorin A,B

(a,d) antibody
Produced in mouse, Clone E3, Ascites fluid, Liquid
G 7775
Monoclonal Anti-Glycophorin C
(b) antibody
Produced in mouse, Clone E5 or 2C10, Whole antiserum, Liquid
B 9277
Monoclonal Anti-Band 3 antibody
Produced in mouse, Clone BIII-136, Ascites fluid, Liquid
R 4011
Monoclonal Anti-Red Cell Wrb

Antigen antibody
Produced in mouse, Affinity purified, One unit will bind 1.0 mg of d-biotin
binds to Wrb, a composite blood group antigen resulting from the association between
Glycophorin A and Band 3
A 8706
Avidin
from egg white, recombinant
Affinity purified, One unit will bind 1.0 mg of d-biotin
B 8041
Biglycan
Essentially salt-free, lyophilized powder, sterile-filtered

Interacts with collagen type I, as well as with fibronectin and TGF-b.
C 1063
Chorionic gonadotropin human
Lyophilized powder, sterile-filtered

vial of 2,500 I.U.
C 5297
Chorionic gonadotropin human
Lyophilized powder, Potency: approx. 3,000 I.U. per mg
G 4877
Gonadotropin
from pregnant mare serum
1,500-6,000 I.U./mg, PMSG
C 0755
Conalbumin chicken egg white

(Ovotransferrin)
Substantially iron-free, Minimum 98%
D 8428
Decorin
Salt-free, lyophilized powder, sterile-filtered

Decorin interacts with collagen type I and II, fibronectin, thrombospondin, and TGF-b.
F 2379
Fetuin
Lyophilized powder (from sodium acetate buffer)

free N-acetylneuraminic acid approx. 0.2%
F 3004
Fetuin
Lyophilized powder
Further processing of F 2379 by gel filtration.
F 3879
Fibrinogen
from human plasma
Type I, Contains approx. 15% sodium citrate and approx. 25% sodium chloride.
Approx. 60% protein (80-90% of protein clottable)
F 4385
Fibrinogen
from murine plasma
Contains approx. 20% sodium citrate and approx. 30% sodium chloride.
Approx. 50% protein (over 80% of protein clottable)
F 8630
Fibrinogen
from bovine plasma
Type I-S powder, powder containing approx 75% protein, approx. 10% sodium citrate
and approx. 15% sodium chloride
F 4883
Fibrinogen
from human plasma
Essentially plasminogen-free powder containing approx. 50% protein, approx. 20% sodium
citrate and approx. 30% sodium chloride
H 7017
Hemocyanin Megathura crenulata

(keyhole limpet)
Lyophilized powder, contains stabilizing buffer, mol wt 8,000-9,000 kDa

vial of 20 mg KLH (in approximately 100 mg total weight)
H 8283
Hemocyanin Megathura crenulata

(keyhole limpet)
in PBS solution, Chromatographically purified

Concentration 3-7 mg/ml protein (A280)
K 3009
Keratan sulfate proteoglycan

from bovine cornea
sterile-filtered
L 0520
Lactoferrin
from human milk
approx. 90% (SDS-PAGE) Lyophilized powder

Chromatographically purified. Contains sodium chloride
L 2020
Laminin
from Engelbreth-Holm-Swarm
murine sarcoma
1 mg/ml in Tris buffered NaCl cell culture, tested

sterile-filtered
M 1778
Mucin
from porcine stomach
Type III partially purified powder, Bound sialic acids, approx. 1%
M 3895
Mucin
from bovine submaxillary glands
Type I-S Lyophilized powder, Neuraminidase substrate

Bound sialic acids approx. 12%
sigma-aldrich.com
A 9791
20
sigma-aldrich.com

P 6782
Peroxidase horseradish
Essentially salt-free, Lyophilized powder 250-330 units/mg solid (using pyrogallol) Type VI-A
P 8375
Peroxidase horseradish
Type VI Essentially salt-free, lyophilized powder 250-330 units/mg solid
P 5661
Plasminogen
from human plasma
e-Aminocaproic acid free, Lyophilized powder containing NaCl, EDTA, lysine, and Tris buffer,
plasmin <0.001 unit/unit plasminogen
T 1001
Thyroglobulin
from bovine thyroid
Powder, Electrophoretically heterogeneous
T 1408
holo-Transferrin bovine
approx. 98%, iron-saturated
T 4132
holo-Transferrin human
minimum 98%, iron-saturated
21

Analysis of the glycan structure of glycoproteins normally requires enzymatic or chemical
methods of deglycosylation.
sigma-aldrich.com
Chemical Deglycosylation
Strategies
Removal of carbohydrates from glycoproteins is useful for a number of reasons:

To simplify analysis of the peptide portion of the glycoprotein
To simplify the analysis of the glycan component
To remove heterogeneity in glycoproteins for X-ray crystallographic analysis
To remove carbohydrate epitopes from antigens
To enhance or reduce blood clearance rates of glycoprotein therapeutics
To investigate the role of carbohydrates in enzyme activity and solubility
To investigate ligand binding
For quality control of glycoprotein pharmaceuticals
22

Hydrazinolysis
Strategies
Hydrazine hydrolysis has been found to be effective in

the complete release of unreduced O- and N-linked
oligosaccharides. Selective and sequential release of
oligosaccharides can be accomplished by initial mild
hydrazinolysis of the O-linked oligosaccharides at 60 C
followed by N-linked oligosaccharides at 95 C. Hydrazine
hydrolysis leaves the glycan intact but results in destruction of the protein component.
Glycan release is accomplished by the addition of fresh
anhydrous hydrazine to a salt-free, freshly lyophilized
glycoprotein sample. The volume of hydrazine should
produce a protein concentration of 5 to 25 mg/ml. The
reaction mixture should be capped immediately. For the
release of both N- and O-linked glycans incubation should
be for 4 hours at 95 C. For the selective release of
O-linked glycans, incubation at 60 C for 5 hours is suitable.
Excess unreacted hydrazine can be removed under high
vacuum at a temperature not exceeding 25 C. The vacuum
pump should be equipped with an activated charcoal/
alumina trap. Addition of small aliquots of anhydrous
toluene may be required to bring the sample to complete dryness.
The dried sample should then be re-N-acetylated on ice
by the addition of ice-cold saturated sodium bicarbonate
solution, followed immediately by the addition of acetic
anhydride. The sample is mixed gently and incubated at
room temperature for 10 minutes. A second equal aliquot
of acetic anhydride is added and incubated for an additional
20 minutes. A 5-fold molar excess of acetic anhydride
over the amine content of the protein should be used.
The volume of sodium bicarbonate added should yield
a 0.5 M acetic anhydride final concentration in the reaction mixture.
A small amount of the released glycan pool may exist as

the acetohydrazide derivative. These derivatives may be
converted to the unreduced glycans by resuspension of
the dryed glycan pool in 1 mM Cu(II) acetate in 1 mM
acetic acid and incubation at room temperature for
one hour.
Dowex 50WX2 can be used to remove the cations and
the sample is washed through with water.
Glycan and protein components can be separated by gel
filtration or by paper chromatography.
Complete details for this procedure are given in Methods
in Enzymology. Care should be taken during all steps of
this procedure as many of the reagents and reactions are
extremely hazardous and highly reactive.
References
1. Patel, T., Bruce, J., Merry, A.H., Bigge, J.C., Wormald, M.R., and
Parekh, R.B., Biochemistry, 32, 679-693 (1992).
2. Takasaki, S., and Kobata, A., Meth. Enzymol., 50, 50-54 (1978).
Key Products
H 2761
Hydrazine
24,451-1
Toluene, Anhydrous
S 6297
Sodium Bicarbonate
A 6404
Acetic Anhydride
C 5893
Cupric Acetate
21,747-6
Dowex 50WX2-400
Peptide
CH2OH
R
O
CH2OH
H
N
O
HO
C
NH
H3C
NH
CH2
O
CH
C
NH2
NH
Hydrazinolysis
C
H3C
Peptide
H
N
O
HO
C
O
sigma-aldrich.com
Re-N-acetylation
CH2OH
R
O
CH2OH
O
HO
NH
H3C
C
O
OH
Regeneration of
reducing
terminus
O
HO
C
NH
NH
H3C
H
N
C
O
CH3
23
Alkaline b-Elimination
Trifluoromethanesulfonic Acid
Release of glycans by alkaline b-elimination utilizes ammonium hydroxide/carbonate or sodium hydroxide (in conjunction with sodium borohydride).
Trifluoromethanesulfonic acid (TFMS) hydrolysis leaves an

intact protein component, but results in destruction of
the glycan.
O-Glycosidic linkages between glycans and the b-hydroxyl

groups of serine or threonine are readily hydrolyzed by
dilute alkaline solutions (0.05 to 0.1 M sodium hydroxide
or potassium hydroxide) under mild conditions (45 to 60 C
for 8 to 16 hours) leading to the liberation of O-glycans
by the mechanism of b-elimination. To prevent isomerization or degradation of the carbohydrates by peeling
reactions, the hydrolysis is performed in the presence of
a reducing agent (0.8 to 2 M sodium borohydride). This
results in the formation of the reduced (alditol) forms of
the glycans. N-Linked glycans are not cleaved under these
conditions, and neither are O-glycans attached to tyrosine,
hydroxyproline, and hydroxylysine. Furthermore, the
b-elimination reaction does not take place if the glycan
is attached to serine or threonine at the carboxy-terminus
of the protein.
Glycoproteins from animals, plants, fungi, and bacteria

have been deglycosylated by this procedure. It has been
reported that the biological, immunological, and receptor
binding properties of some glycoproteins are retained
after deglycosylation by this procedure, although this
may not be true for all glycoproteins. The reaction is
non-specific, removing all types of glycans, regardless of
structure, although prolonged incubation is required for
complete removal of O-linked glycans. Also, the innermost Asn-linked GlcNAc residue of N-linked glycans
remains attached to the protein. The method removes
the N-glycans of plant glycoproteins that are usually
resistant to enzymatic hydrolysis.
For quantitative release of N-linked glycans, harsher

alkaline conditions are required (1 M sodium hydroxide
at 100 C for 6 to 12 hours). Again, the reaction must be
performed under reducing conditions (1 to 2 M sodium
borohydride) to prevent peeling reactions taking place
on the released N-glycans. N-Acetylglucosamine (GlcNAc)
residues are de-acetylated during this reaction and must
be re-N-acetylated (acetic anhydride in methanol) during
the recovery of the glycans.
Strategies
References
1. Patel, T.P., and Rarekh, R.B., Meth. Enzymol., 230, 57-66 (1994).
2. Bendiak, B. and Cumming, D.A., Carbohydr. Res., 151, 89 (1986).
3. Makino, Y., et al., J. Biochem., 128, 175-180 (2000).
4. Piller, F., and Piller, V., in Glycobiology: A Practical Approach,
Fukuda, M. and Kobata, A. (Eds), pp. 291-328 (IRL/Oxford Univ.
Press, (Oxford UK 1993)).
5. Burgess, A.J., and Norman, R.I., Eur. J. Biochem. 178, 527-533
(1988).
6. Edge, A.S.B., J. Biochem., 376, 339-350 (2003).
7. Sojar, H.T., and Bahl, O.P., Meth. Enzymol, 138, 41-350 (1987).
8. Edge, A.S.B., et al., Anal. Biochem., 118, 131-137 (1981).
References
1. Carlson, D.M., and Blackwell, C., J. Biol. Chem., 243, 616-626 (1968).
2. Lloyd, K.O., Burchell, J., Kudryashov, V., Yin, B.W.T., and TaylorPapadimitriou, J., J. Biol. Chem., 271, 33325-33334 (1996).
3. Morelle, W., Guyetant, R., and Strecker, G., Carbohydr. Res., 306,
435-443 (1998).
GlycoProfile IV Chemical
Deglycosylation Kit
Product Code PP0510
The GlycoProfile Chemical

Deglycosylation Kit has been optimized to provide a rapid, convenient, and reproducible method to
remove glycans from glycoproteins by
reaction with trifluoromethanesulfonic acid
(TFMS). The deglycosylated protein can then be recovered
using a suitable downstream processing method. The kit
contains sufficient reagents and a glycoprotein standard,
for a minimum of 10 reactions when the sample size is
between 1 and 2 mg of a typical glycoprotein. Unlike other
chemical deglycosylation methods, hydrolysis with anhydrous TFMS is very effective at removing O- and N-linked
glycans (except the innermost Asn-linked GlcNAc or
GalNAc) and results in minimal protein degradation.
Visit
sigma-aldrich.com/glycogo
to find out more about the latest
glycobiology products
from Sigma-Aldrich
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24

Sequential hydrolysis of individual monosaccharides from glycans can be useful for the
elucidation of the structure and function of the glycan component. Due to the restraints
of the specificity of glycolytic enzymes currently available, sequential hydrolysis of individual
monosaccharides is also required in many instances in order to completely remove a glycan
component enzymatically. This is particularly true in the enzymatic deglycosylation of
many O-linked glycans.
N-Linked Glycan Strategies
Enzymatic Deglycosylation
Strategies
Use of the enzyme PNGase F is the most effective method

of removing virtually all N-linked oligosaccharides from
glycoproteins. The oligosaccharide is left intact and, therefore, suitable for further analysis (the asparagine residue
from which the sugar was removed is deaminated to
aspartic acid, the only modification to the protein). A
tripeptide with the oligosaccharide-linked asparagine as
the central residue is the minimal substrate for PNGase F.
However, oligosaccharides containing a fucose a(1-3)-linked
to the asparagine-linked N-acetylglucosamine, commonly
found in glycoproteins from plants or parasitic worms,
are resistant to PNGase F. N-Glycosidase A (PNGase A),
isolated from almond meal, must be used in this situation.
This enzyme, however, is ineffective when sialic acid is
present on the N-linked oligosaccharide. Other commonly
used endoglycosidases such as Endoglycosidase H and

the Endoglycosidase F series are not suitable for general
deglycosylation of N-linked sugars because of their limited
specificities and because they leave one N-acetylglucosamine
residue attached to the asparagine.
Steric hindrance slows or inhibits the action of PNGase F
on certain residues of glycoproteins. Denaturation of the
glycoprotein by heating with SDS and 2-mercaptoethanol
greatly increases the rate of deglycosylation.
Through sequential deglycosylation of monosaccharides,
all complex oligosaccharides can be reduced to the trimannosyldiacetylchitobiose core by selective hydrolysis with
a neuraminidase, b-galactosidase, and N-acetylglucosaminidase, available as part of the Enzymatic Deglycosylation
Kit (Product Code E-DEGLY). Fucosidases may be required
in some situations.
PNGase F
sigma-aldrich.com
R = N- and C-substitution by groups other than H

R = H or the rest of an oligosaccharide
R3 = H or a(1-6)fucose
25
For some glycoproteins, no cleavage by PNGase F occurs

unless the protein is denatured. For others, some or all
of the oligosaccharides can be removed from the native
protein after extensive incubation of three days or longer.
PNGase F will remain active under reaction conditions
for at least three days allowing extended incubations of
native glycoproteins. In general, it appears that particular
residues, due to their location in the native protein structure, are resistant to PNGase F and can not be removed
unless the protein is denatured.
Endoglycosidases F1, F2, and F3 are less sensitive to protein
conformation than PNGase F and are more suitable for
deglycosylation of native proteins. Sigmas Native Protein
Deglycosylation Kit (Product Code N-DEGLY) supplies all
three of these enzymes with reaction buffers and detailed

interactions. The linkage specificities of Endoglycosidases
F1, F2, and F3 suggest a general strategy for deglycosylation of proteins that may remove all classes of N-linked
oligosaccharides without denaturing the protein. Initially
complex oligosaccharides can be reduced to the trimannosyldiacetylchitobiose using neuraminidase, b-galactosidase,
and N-acetylglucosaminidase. Fucosidases may be
required in some situations. The remaining trimannosyldiacetylchitobiose core structures can be removed with
Endoglycosidase F3. Bi- and triantennary structures can
be immediately removed by Endoglycosidases F2 and
F3, respectively.
Strategies
Native and Sequential N-Linked

Glycan Strategies
High-mannose (oligomannose) and hybrid structures can

be removed by Endoglycosidase F1, but not complex
oligosaccharides.
High-Mannose Glycans
sigma-aldrich.com
Hybrid Glycans
26

Endo F2 and Endo F3 have the ability to cleave complex
structures. Endo F2 cleaves biantennary complex and to
lesser extent high mannose oligosaccharides. Fucosylation
has little effect on Endo F2 cleavage of biantennary
structures. Endo F2 will not cleave hybrid structures.
Endo F3 cleaves biantennary and triantennary complex
oligosaccharides. However, non-fucosylated biantennary
and triantennary structures are hydrolyzed at a slow rate
by Endo F3. Core fucosylated biantennary structures are
efficient substrates for Endo F3 oligosaccharides. Core
fucosylation of biantennary structures increases activity

up to 400-fold. Endo F3 has no activity on oligomannose
and hybrid molecules.
Endo F3 will cleave fucosylated and non-fucosylated trimannosyl core structures on free and protein-linked glycans.
Native deglycosylation of complex tetraantennary glycans
requires sequential hydrolysis down to the trimannosyldiacetylchitobiose core.
sigma-aldrich.com
Strategies
Biantennary Glycans
27
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Strategies
Triantennary Glycans
28
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Strategies
Complex Tetraantennary Glycans
29
There is no enzyme comparable to PNGase F for removing

intact O-linked sugars. Monosaccharides must be sequentially hydrolyzed by a series of exoglycosidases until only
the Gal-b(1-3)-GalNAc core remains. O-Glycosidase can
then remove the core structure intact with no modification of the serine or threonine residue. Denaturation of
the glycoprotein does not appear to significantly enhance
O-deglycosylation. Any modification of the core structure
can block the action of O-Glycosidase. The most common
modification of the core Gal-b(1-3)-GalNAc is a mono-, di-,
or tri-sialylation. These residues are easily removed by
a(2-3,6,8,9)-Neuraminidase since only this enzyme is capable of efficient cleavage of the NeuNAc-a(2-8)-NeuNAc
bond. Another commonly occurring O-linked hexasaccharide
structure contains b(1-4)-linked galactose and b(1-6)-linked
N-acetylglucosamine as well as sialic acid. Hydrolysis of
this glycan will require, in addition to neuraminidase, a

b(1-4)-specific galactosidase and an N-acetylglucosaminidase.
A non-specific galactosidase will hydrolyze b(1-3)-galactose
from the core glycan and leave O-linked GalNAc that cannot
be removed by O-Glycosidase. b(1-4)-Galactosidase and
b-N-acetylglucosaminidase can be used for the hydrolysis
of these and any other O-linked structures containing
b(1-4)-linked galactose or b-linked N-acetylglucosamine
such as polylactosamine. Less common modifications that
have been found on O-linked oligosaccharides include
a-linked galactose and a-linked fucose. Directly O-linked
N-acetylglucosamine (found on nuclear proteins) and
a-linked N-acetylgalactosamine (found in mucins) have
also been reported. Additional exoglycosidases are necessary for complete O-deglycosylation when these residues
are present. Fucose and mannose directly O-linked to
proteins cannot presently be removed enzymatically.
Strategies
O-Linked Glycan Strategies
Di- and Trisialated O-Linked Glycans
sigma-aldrich.com
O-Linked Core 2 Hexasaccharide
30

Deglycosylation Kits
GlycoProfile I
Enzymatic In-Gel N-Deglycosylation Kit
Strategies
Product Code PP0200
R1 = N- and C-substitution by groups other than H

R2 = H or the rest of an oligosaccharide structure
In-gel Deglycosylation
Excise protein
of interest
Place gel slice in
siliconized tube
Destain gel slice
Remove destaining
solution and dry
gel in Speed Vac
Rehydrate with
PNGase F solution
Incubate for
30 min. at 37 C
Add water
Incubate 2 hours to
overnight at 37 C
Remove glycancontaining supernatant.
Analyze if desired.
Glycosylation often leads to problems in subsequent protein analysis procedures. Glycopeptides generally do not
readily ionize during MS analysis leading to insufficient
spectral data. Furthermore, proteolytic digestion of the
native glycoprotein is often incomplete due to steric
hindrance by the oligosaccharides. Removal of the
carbohydrate groups from a glycoprotein prior to
protein identification is preferred.
Sigmas GlycoProfile I Enzymatic In-gel N-Deglycosylation
Kit is optimized to provide a convenient and reproducible
method to N-deglycosylate and tryptically digest protein
samples in 1D or 2D polyacrylamide gel slices for subsequent MS or HPLC analysis. The procedure is suitable for
Coomassie Blue and Colloidal Coomassie stained gels and
may be used with gels silver stained and destained using
Sigmas Proteo Silver Plus Kit (Product Code PROT-SIL2).
GlycoProfile Enzymatic In-gel N-Deglycosylation kit includes
the enzymes and reagents necessary for N-linked deglycosylation and tryptic digestion. The samples can then be
desalted and concentrated for analysis by MALDI-TOF or
electrospray MS.
Features & Benefits
Provides all components for in-gel deglycosylation and
trypsinization of protein samples Conveniently prepares deglycosylated protein samples for analysis by
MS or HPLC
Utilizes PNGase F for the enzymatic removal of N-linked
glycans Proteins remain intact, unlike the use of chemical deglycosylation which can degrade the protein
Includes Proteomics Grade PNGase F and Trypsin
Highly purified enzymes possess no unwanted activities
or additives to complicate analysis
PNGase F is supplied lyophilized from a low salt buffer
Allows reconstitution of the enzyme to any concentration needed
Works in solution or with gel slices Allows choice
of methods
Components
Destaining Solution
Proteomics Grade PNGase F
Wash gel slice

with water
sigma-aldrich.com
Remove and
discard liquid
Proteomics Grade Trypsin

Trypsin Solubilization Reagent
Trypsin Reaction Buffer
Invertase (positive control)
Peptide Extraction Solution
Dry gel slice in

Speed Vac
Sample is now ready to be

rehydrated in Trypsin solution
(see Trypsin In-Gel Digest kit).
Biotech Grade Acetonitrile
31

GlycoProfile II
Enzymatic In-Solution N-Deglycosylation Kit
The GlycoProfile Enzymatic In-Solution N-Deglycosylation
Kit has been optimized to provide a convenient and reproducible method to remove N-linked glycans from glycoproteins and is compatible with subsequent MALDI-TOF
mass spectrometric analysis without interference from
any of the reaction components. The kit contains sufficient enzyme, glycoprotein standard and reagents, for
a minimum of 20 reactions when the sample size is
between one to two mg of a typical glycoprotein.
Features & Benefits
Provides all components for in-solution N-linked deglycosylation of protein samples Conveniently prepares
deglycosylated protein samples for analysis by MS,
HPLC and PAGE
Reagents are optimized for direct MS analysis No need
for post-reaction sample clean up
Utilizes PNGase F for the enzymatic removal of N-linked
glycans Proteins remain intact, unlike the use of chemical deglycosylation which can degrade the protein
Includes Proteomics Grade PNGase F and Trypsin
Highly purified enzymes possess no unwanted activities
or additives to complicate analysis
PNGase F is supplied lyophilized from a low salt buffer
Allows reconstitution of the enzyme to any concentration needed
Components
Strategies
Product Code PP0201

In-solution Deglycosylation
Aliquot glycoprotein sample
Add denaturant
Incubate at 100 C for 10 min
Cool to room temperature
Proteomics Grade PNGase F

Ribonuclease B
10x Reaction Buffer
Add reaction buffer and PNGase F
Octyl b-D-Glucopyranoside
Incubate at 37 C for 1 hour
Analyze by MS, PAGE or HPLC
sigma-aldrich.com
2-Mercaptoethanol
32

Key Endoglycosidases
PNGase F
Synonyms: Glycopeptidase F; N-Glycosidase F; Peptide
N-Glycosidase F; Peptide-N4-(acetyl-b-glucosaminyl)asparagine amidase
EC 3.5.1.52
Molecular Weight: 36 kDa
Strategies
PNGase F cleaves all asparagine-linked complex, hybrid,

or high mannose oligosaccharides unless b(1-3) core
fucosylated. The asparagine must be peptide bonded
at both termini. The asparagine residue from which the
glycan is removed is deaminated to aspartic acid.
Detergent and heat denaturation increases the rate of
cleavage up to 100 times. Most native proteins can still
be completely N-deglycosylated, but incubation time must
be increased. The optimal pH is 8.6 and the enzyme is
active in the pH range of 6 to 10.

CH2OH
R
O
NH
Peptide
CH2OH
R
O
O
HO
PNGase F
NH
CH2
C
NH
H3C
H
N
OH
HO
H3C
Peptide
CH
C
O
NH
CH2
C
O
Peptide
HO
CH
Core GlcNAc
Asparagine Residue
Peptide
Aspartic Acid Residue

SDS-PAGE analysis of RNase B treated with varying amounts of PNGase F Enzyme
PNGase F Products
P 7367
8
PNGase F
Synonyms: N-Glycanase, N-Glycosidase F, Proteomics Grade

Features and Benefits
Excellent for applications requiring N-linked deglycosylation.
Superior performance in on-blot, in-gel, and in-solution digestion methods.
High activity, minimum 25,000 units per milligram.
Compatible for use in MALDI-TOF Mass Spectroscopy.
Each lot tested for suitability.
sigma-aldrich.com
Proteomics Grade PNGase F from Sigma-Aldrich is extensively purified and contains minimal
residual buffer salts and no glycerol or other stabilizers or preservatives that may interfere
in sensitive glycoprotein analysis methods.
Lane 1: Control (no PNGase F)

Lane 2-10: Test (+ 0.01 to 0.09 U PNGase F)
G 5166
8
PNGase F
Synonyms: N-Glycanase, N-Glycosidase F

from Chryseobacterium (Flavobacterium), Buffered Aqueous Solution
33

Deglycosylation Products
P 9120
8
PNGase F
Synonyms: N-Glycanase, N-Glycosidase F

Recombinant, Solution
N 3786
a(23,6,8,9) Neuraminidase
Synonyms: Sialidase, Acyl-neuraminyl Hydrolase, Proteomics Grade

from Arthrobacter ureafaciens
PP0200*
8
Glycoprofile I
In-Gel N-Deglycosylation Kit
Excellent performance when used for in-gel N-linked deglycosylation of glycoproteins

and glycopeptides. Utilizes extensively purified Proteomics Grade PNGase F and Trypsin.
Compatible with MS (TOF or Electrospray) analysis following desalting. Sufficient for a
minimum of 10 samples.
PP0201*
8
Glycoprofile II
In-Solution N-Deglycosylation Kit
Superior, reproducible method to remove N-linked glycans from glycoproteins. Utilizes

extensively purified Proteomics Grade PNGase F. Compatible with subsequent MALDI-TOF
mass spectrometric analysis, no need for desalting. Sufficient for a minimum of 20 reactions.
N-DEGLY
Native Deglycosylation Kit
The N-DEGLY kit includes Endoglycosidase F1, F2, and F3 as well as an optimized reaction
buffer for each. The kit is intended for use in the deglycosylation of N-linked oligosaccharides
from glycoproteins under native conditions. Particular residues, due to their location in
the native protein structure, are often resistant to the traditional deglycosylation methods
using PNGase F and cannot be removed unless the protein is denatured. Endoglycosidases
F1, F2, and F3 are less sensitive to protein conformation than PNGase F and are more suitable for deglycosylation of native proteins.
Strategies
Features and Benefits

Compatible for use in MALDI-TOP MS applications.
Cleaves all non-reducing, unbranched NANA/NeuNAc and NeuGc residues.
Ideal for complete, non-specific removal of sialic acid groups.
*For additional product information, refer to pages 30 and 31.
PNGase A
PNGase A (Glycopeptidase A, Peptide N-Glycosidase A),
hydrolyzes oligosaccharides containing a fucose a(1-3)linked to the asparagine-linked N-acetylglucosamine,
commonly found in glycoproteins from plants or parasitic
worms. These types of glycans are resistant to PNGase F.
Like PNGase F the asparagine asparagine residue from
which the glycan is removed is deaminated to aspartic
acid. However, it is ineffective when sialic acid is present
on the N-linked oligosaccharide.

G 0535
PNGase A
Buffered solution, min. 0.5 units/ml
G 1163
O-Glycosidase
O-Glycosidase hydrolyzes the serine- or threonine-linked unsubstituted O-Glycan core

[Gal-b(1-3)-GalNAc]. Any modification of the core structure can block the action of
O-Glycosidase.
E-DEGLY
Enzymatic Deglycosylation Kit
Completely removes all N- and simple O-linked carbohydrates from glycoproteins.

Efficiently digest polysialylated carbohydrates. Simplifies amino acid sequencing applications.
Removes carbohydrate epitopes from antigens. Native & denaturing procedures included
in bulletin.
sigma-aldrich.com
Deglycosylation Products
34

Endoglycosidase F1, F2 & F3
Strategies
Endoglycosidase F1 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose
glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine
residue remaining on the asparagine. High mannose
(oligomannose) and hybrid structures can be removed
by Endoglycosidase F1, but not complex, oligosaccharides.
Useful under native or non-denaturing deglycosylation
conditions.
residue remaining on the asparagine. Endo F3 cleaves
non-fucosylated biantennary and triantennary structures
at a slow rate, but only if peptide-linked. Core fucosylation of biantennary structures increases activity up to
400-fold. Endo F3 has no activity on oligomannose and
hybrid molecules. Endo F3 will also cleave fucosylated
trimannosyl core structures on free and protein-linked
oligosaccharides. Native deglycosylation of complex
tetrantennary glycans requires sequential hydrolysis
down to the trimannosyl-diacetylchitobiose core.
R1 = Asn or H
R2 = Oligomannose or hybrid configurations
residue remaining on the asparagine. Endo F2 cleaves
biantennary complex and to a lesser extent high mannose oligosaccharides. Fucosylation has little effect on
Endo F2 cleavage of biantennary structures. Endo F2 will
not cleave hybrid structures. Useful under native or nondenaturing deglycosylation conditions.

R2 = Biantennary and triantennary complex oligosaccharides or
trimannosylchitobiose core
R4 = Asn (H or Asn if core fucosylated)
R1 = H or Asn
R2 = Biantennary and oligomannose configurations
sigma-aldrich.com
Endoglycosidase F Products
E 9762
Endoglycosidase F1
Solution, from Chryseobacterium (Flavobacterium) meningosepticum
E 0639
Endoglycosidase F2
E 2264
Endoglycosidase F3
35

Endoglycosidase H cleaves between the N-acetylglucosamine residues of the chitobiose core of N-linked glycans,
leaving one N-acetylglucosamine residue attached to the
asparagine. The specificity of this enzyme is such that
oligomannose and most hybrid types of glycans, including
R1
R2
R3
R4
=
=
=
=
those that have a fucose residue attached to the core

structure, are cleaved whereas complex type glycans are
not released. Thus this enzyme is extremely useful for
selective release of oligomannose or hybrid type glycans
from glycoproteins. The enzyme is also active against
dolichol-linked glycans containing these structures.
Strategies
Endoglycosidase H
Oligomannose (2-150)
H or mono- or oligosaccharide at the C2 or C4 position
H or a(1-6)fucose
Asn or Dolichol pyrophosphate
The enzyme has a molecular weight of approximately

27 kDa. The workable pH range is between 5.0 and 6.0,
with the optimal pH at 5.5. No loss of activity is observed
during incubation at 37 C for 48 hours over the pH
range 4.5 to 8.5. However, below pH 4.5, activity is
rapidly lost.
Endoglycosidase H
From recombinant with reaction buffer
E 6878
Endoglycosidase H
From Streptomyces griseus
E 7642
Endoglycosidase H
Recombinant, from Streptomyces plicatus, expressed in E. coli
sigma-aldrich.com
Endoglycosidase H Products
A 0810
36
Glycan Analysis and Labeling

In contrast to proteins and peptides, glycans
do not absorb ultraviolet (UV) light strongly,
thereby giving a weak detector signal, even
at 214 nm. Furthermore, as glycans with
various different structures may be present
in minute amounts in glycoprotein hydrolysates, their detection by UV absorbance
may not be practical. As a result a wide
range of alternative techniques have been
developed for the detection of glycans.
Glycan Analysis
and Labeling
Instead of UV detectors for on-line HPLC

system, refractive index and pulsed amperometric detectors, with sensitivity in the
range of 10 to 100 pmol, have enabled
improved detection of native mono- and
oligosaccharides. Other approaches to
improve detection include labeling glycans
with radioactive isotopes, but this technique is not widely used due to the safety
requirements for the handling of radiolabeled substances.
Most glycoproteins exist as a heterogeneous
population of glycoforms or glycosylated
variants with a single protein backbone and
a heterogeneous population of glycans at
each glycosylation site. It has been reported

in the literature that for some glycoproteins,
100 or more glycoforms exist at each glycosylation site. In view of this heterogeneity
and the presence of branched structures,
the analysis of glycans is much more complicated than protein chemistry. It requires
several different strategies to separate and
study the structure of each individual glycan.
Once the glycans have been released from
the glycoprotein, the glycan pool can be
analyzed by MALDI-TOF mass spectrometry
or, after fluorescent labeling, by either HPLC
or MS, or both. This strategy can provide
a glycan profile or a glycosylation pattern that is highly characteristic of the
glycoprotein. The technology can be applied
to compare glycan profiles of glycoproteins
found in normal and diseased states, or to
compare different batches of recombinant
protein products. Both these techniques
provide valuable information in terms of
composition, linkage and arm specificity
(using various exoglycosidases) from which
structural information on individual glycans
can be elucidated.
Glycan Labeling
Chemical derivatization is now the most common method
used for labeling glycans at their reducing ends by reductive amination. A single molecule of fluorescent label can
be incorporated to each mono- or oligosaccharide, thus
allowing molar quantities to be determined. The sensitivity
Technique
HPLC
sigma-aldrich.com
Gel Electrophoresis
of detection by this technique is in the low femtomole

range and depending upon the analytical technique, an
appropriate fluorescent label can be used. The table below
lists examples of some of the fluorophores widely used
in glycan analysis.
Fluorescent Label
Abbreviation
Product Code
2-Aminobenzoic acid
2-AA
10678
2-Aminobenzamide
2-AB
10710
2-Aminopyridine
2-AP
09340
8-Aminonaphthalene-1,3,6-trisulfonic acid
ANTS
08658
2-Aminoacridone
AMAC
06627
Capillary Electrophoresis
9-Aminopyrene-1,3,6-trisulfonic acid
APTS
09341
Mass spectrometry
2-Aminobenzamide
2-AB
10710
37

HPLC Analysis of Glycans
Fetuin
16,000
12,000
8,000
4,000
0
70
80
90
100
110
120
130
Retention time (minutes)

Figure 2. HPLC Profile of the 2-AB labeled N-linked glycan library
obtained from Fetuin.
Partially hydrolyzed dextran, as a source of glucose oligomers, is used as an external standard and the elution
position of each peak is expressed in glucose units (gu)
(see Fig. 3). The elution positions of peaks in an unknown
glycan pool are assigned an overall gu value by comparison with the standard dextran ladder. These values may
then be used to predict possible structures for unknown
glycans. For example, it has been reported that the glycans
released from RNase B consist solely of N-linked oligomannose structures. The five peaks obtained upon profiling
of the glycans from RNase B can therefore be assigned
structures ranging from Man-5 to Man-9, as indicated
on Figure 1.
1
4
5
4,400
800
Glycan Analysis
and Labeling
Normal phase chromatography

The column used in this mode of chromatography consists
of a matrix with imide or amide functional groups. Samples
are applied in an organic solvent solution to the column
are then eluted with increasing concentration of aqueous
buffer. This exploits the subtle differences in hydrophilicity
between individual glycans and their affinity for the
column matrix, thereby achieving high resolution and
reproducibility. The ability to analyze sialylated and neutral
sugars in one chromatographic run makes it a useful technique for profiling both N- and O-glycan pools, and for
making comparisons between different glycan samples.
Figures 1 and 2 show the elution profiles of N-linked
glycans released from two model glycoproteins, RNase B
and Fetuin, respectively.
20,000
Fluorescence
The released glycan pool from glycoprotein hydrolysis

can be separated into components depending upon the
characteristics of the glycans and the HPLC matrices used.
Three types of chromatography, normal phase, weak anion
exchange, and reversed phase HPLC, have been generally
used with fluorescent-labeled oligosaccharides for the
separation of sialylated and neutral N- and O-linked
glycans. High pH anion exchange chromatography with
pulsed amperometric detector can be used with native
glycans, but has a major drawback in that the separation
is achieved in high molarity sodium hydroxide that has
to be removed to recover glycans for further structural
analysis.
RNase B
Fluorescence
Man-5
Fluorescence
600
Man-6
400
3,300
7
2,200
8
9
10
11
1,100
Man-8
200
Man-7
Man-9
0
70
80
90
100
110
120
20
40
60
80
100
120
140
160

Figure 3. Separation of partially hydrolyzed 2-AB labeled dextran on
normal phase HPLC. The numbers indicate glucose units (gu).
sigma-aldrich.com
Figure 1. HPLC Profile of the 2-AB labeled N-linked glycan library

obtained from RNase B.
38
Glycan Analysis
and Labeling
In some of these charge classes, for example in the

disialylated glycan peak area shown in Figure 4(A), the
partially-resolved peak contains bi- and triantennary
structures with the triantennary glycans eluting first.
The peaks corresponding to each charge band can be
collected, concentrated, further separated, and analyzed
by normal phase chromatography. Two examples are
shown in Figures 4(B) and 4(C).
A)
Fetuin glycan
library
Fluorescence
8,000
6,000
Neutral
Di
Tetra
Tri
4,000
Mono
2,000
0
0
10
12
14
16
18
20
22

B)
400
Neutral glycans
Fluorescence
300
200
100
sigma-aldrich.com
0
60
70
80
90
100
110
120
130
140
C)
400
Monosialylated glycans
300
Fluorescence
Weak anion exchange chromatography

Weak anion-exchange chromatography relies upon the
relative binding affinities of the glycans to the weak anion
exchange matrix in the column. This type of chromatography separates N- and O-linked glycans on the basis of
the number of charge groups they contain. The elution
position is therefore determined by the number of sialic
acid, sulfate, phosphate, or uronic acid moieties present
on the glycan, but also to some extent by the size of the
glycan. Thus, within one charge band, larger structures
elute before smaller ones. For example, Figure 4(A) shows
the separation of the N-linked glycan library released
from fetuin into five major peaks. The flow-through peak
contains neutral glycans, while peaks eluting with increasing time represent the mono-, di-, tri- and tetrasialylated
glycans of fetuin.
200
100
0
60
70
80
90
100
110
120
130
140

Figure 4. Separation of neutral and acidic glycans of fetuin by weak
anion-exchange chromatography. (A) The separation of the glycan
pool into neutral, mono-, di-, tri- and tetrasialylated glycans.
(B) Further separation of the neutral glycan fraction from (A) by
normal phase chromatography. (C) Further separation of the monosialylated glycan fraction from (A) by normal phase chromatography.
Reversed phase chromatography

Reversed phase chromatography separates sugars on the
basis of hydrophobicity and can be carried out on a C18
column. This method is complementary to the normal phase
chromatography, allowing some glycans that co-elute
on one system to be resolved by the other system. This
approach is important during structure determination of
the glycan to assess the shift in the retention time upon
treatment with a particular exoglycosidase. The sample is
applied in aqueous buffer to the column and eluted with
increasing concentration of organic solvent. Unlike the
normal phase chromatography, the elution positions of
glycans are measured by comparison with an arabinose
ladder and assigned as arabinose units (au).
39

MS of neutral glycans
Unlike peptides, neutral glycans exhibit low ionization
efficiency and the [M+H]+ ion is therefore not sufficiently
abundant. However, these glycans can be detected as
alkali metal adducts which ionize efficiently. Normally,
[M+Na]+ is the major ion, accompanied by a weaker [M+K]+
ion. Other adducts, such as [M+Li]+ can be generated by
the addition of the appropriate inorganic salt to the matrix.
The inclusion of NaCl in the matrix solvent allows the
glycans to ionize predominantly in the [M+Na]+ form
with little or no [M+K]+ ion formation.
Mass Spectrometry of Glycans

The development of modern mass spectrometry (MS)
equipment with high resolution and mass accuracy has
led to its use in analyzing glycans for both profiling and
structural studies. Unlike HPLC methods, a substantially
larger amount, about 10-20 times, of glycan is normally
required for a single MS spectrum. Matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry is the most widely used MS technique. The
information gained on using MALDI MS is mass weight
and this can be used to assign putative monosaccharide
structures present in a pure oligosaccharide since the mass
of a monosaccharide is measured with a high degree of
accuracy. Like HPLC, MALDI MS can also be used in conjunction with exoglycosidase enzymes for structural and
linkage analysis of glycans.
Different approaches and matrices are used for the analysis of neutral and acidic glycans (those that contain sialic
acid) by MALDI MS. Neutral glycans ionize with reasonable
efficiency using 2,5-dihydroxybenzoic acid (2,5-DHB)
(Product Code D 1942) as the matrix in positive ion mode.
Acidic glycans generally provide poor MALDI spectra with
DHB matrices due to variable losses of sialic acids or carboxyl groups leading to multiple peaks. These glycans
are therefore analyzed in negative ion mode using an
alternative matrix.
Glycan Analysis
and Labeling
Normally, use of the 2,5-DHB matrix is sufficient for most

applications. However, it has been reported that, in some
instances, a 2-3 fold increase in sensitivity can be obtained
with the use of Super 2,5-DHB matrix as it allows for
softer desorption with a reduction in metastable ion
formation. Super 2,5-DHB consists of a mixture (90:10
ratio, wt %) of 2,5-DHB and 2-hydroxy-5-methoxybenzoic
acid, respectively.
Ovalbumin (0.5 mg) was incubated with 7 units of PNGase F
enzyme (Product Code P 7367) at 37 C for 3 hours and
the glycans recovered by ultrafiltration. A third of this
material was dried under vacuum centrifugation and
then analyzed by MALDI-TOF MS. Figure 5 shows the
N-linked glycan profile of ovalbumin.
C
1136.22
100
A
933.20
D
1257.29
H
1745.52
E
1339.30
1152.26
974.16
%
G
1663.48
1298.30
F
1542.41
1419.33
1704.52
1501.41
975.16
J
I
1948.57
1866.59
B
1095.08
990.12
1762.53
991.14
1035.10
1964.66
K
2110.68
L
2151.74
m/z
900
1000
1100
1200
1300
1400
1500
Figure 5. Positive ion MALDI MS of the N-linked glycans from ovalbumin released by treatment with PNGase F. Recorded from 2,5-DHB
1600
1700
1800
1900
2000
2100
2200
2300
as the matrix. The letters indicate identified peaks, given on the

next pages.
sigma-aldrich.com
0
800
1907.65
1558.40
40
sigma-aldrich.com
Glycan Analysis
and Labeling
Corresponding Glycan Structures for MS Profile

A
Refer to Structure Key on pages 2-4
41
sigma-aldrich.com
Glycan Analysis
and Labeling
Corresponding Glycan Structures for MS Profile
42

Glycan Analysis: General MS Protocol
4. Allow to dry at room temperature.
Note: Mass spectra can be acquired at this stage.
However the following steps have been reported to
improve signals by forming an even film of crystals,
which reduces the need to search for sweet spots
on the target.
5. Onto the dried spot, dispense 1 ml of ethanol and
allow the matrix plus analyte mixture to recrystallize
on the target plate.
6. Dry the spot. The target is now ready to acquire
mass spectra.
7. Figure 6 shows representative MALDI-TOF MS traces
obtained for three glycan standards Man-3 glycan,
Man-8 glycan, and NA4 glycan.
Procedure
The following method, commonly referred to as the
dried-droplet method, is based on the original MALDI
experiments and remains the most commonly used
method in the mass spectrometry community.
1. Transfer 3 ml of the appropriate matrix solution
(either DHB or Super DHB at 10 mg/ml) into a 0.5 ml
Eppendorf tube.
2. Add 1 ml of 10 pmol/ml solution of a standard glycan or
an unknown sample solution to the tube containing
the matrix. Vortex to mix.
3. Dispense an aliquot (approximately 1.5 ml) of this
mixture onto the MALDI target plate.
A)
933.32
Glycan Analysis
and Labeling
100
934.32
935.32
936.33
915.16
949.29
937.33
931.11
950.29
m/z
915
920
925
930
935
940
945
950
955
B)
1743.58
100
1744.59
1745.59
%
1746.59
1747.59
1760.56
sigma-aldrich.com
1748.59
0
m/z
1715
1720
1725
1730
1735
1740
1745
1750
1755
1760
1765
1770
1775
43

C)
2394.85
100
2393.85
2395.85
2396.86
%
2397.86
2398.86
2409.82
m/z
2360
2370
2380
2390
2400
2410
2420
2430
2440
Glycan Analysis
and Labeling
2350
Figure 6. MALDI-TOF mass spectra of (A) Man-3 glycan, (B) Man-8 glycan, and (C) NA4 glycan obtained in positive ion reflectron mode using
DHB as matrix. Each of these glycans are detected as the sodium adduct of the molecular ion [M+Na]+.
GlycoMass I Neutral Glycan

Calibration Kit
Product Code PP0301
COMING
SOON!
This kit provides a range of purified

N-linked glycans for the purpose of
testing MALDI mass spectrometers,
regardless of the instrument manufacturer. In addition to the glycans,
two different, high purity, re-crystallized
matrices and a ready-to-use matrix solvent are provided.
This kit provides the necessary reagents and standards for
the analysis of pure glycans, or for profiling and/or structure determination of complex glycan mixtures by MS.
Visit
to find out more about the latest
glycobiology products
from Sigma-Aldrich
GlycoMass I Neutral Glycan Calibration Kit is supplied

with the following items.
Monoisotopic [M]+ (Da) Monoisotopic [M+Na]+ (Da)
Amount Supplied
M 8418
Man-3 Glycan
910.3278
933.3175
1 nmol (approx.)
M 5918
Man-8 Glycan
1720.5919
1743.5817
1 nmol (approx.)
M 8918
NA4 Glycan
2370.8566
2393.8463
1 nmol (approx.)
D 1942
2,5-DHB matrix
2 x 10 mg
D 1817
Super 2,5-DHB matrix
2 x 10 mg
G 2164
Matrix solvent
(10% ethanol + 10 mM NaCl)
10 ml
sigma-aldrich.com
Product Code Product
44

MS of acidic glycans
DHB is the most widely used matrix for the MALDI MS
analysis of glycans. This matrix, however, is not ideal for
acidic glycans (such as sialylated N-linked sugars) as the
detection limit is poor compared to neutral glycans, and
fragmentation can occur with losses of carboxylic and/or
sialic acid groups. In addition, sialylated glycans analyzed
in the positive-ion mode yield a mixture of cation adducts
producing multiple peaks. One approach to enhance sensitivity is to derivatize the glycan, but this may compromise
the absolute quantitation of the glycan. An alternative
way is to choose a different matrix that can overcome the
problem of fragmentation during analysis and provide
an increased detection limit.
Glycan Analysis
and Labeling
Two most widely used matrices for the analysis of acidic

glycans are 6-aza-2-thiothymine and 2,4,6-trihydroxyacetophenone (THAP, Product Code 91928). The former
gives a significant increase in sensitivity over DHB matrix,
but still causes some fragmentation in the negative ion
linear mode. With THAP, the detection limit of analysis

can be at the 10 fmol level, with little or no evidence
of fragmentation in the linear mode. The conditions for
sample preparation with this matrix (outlined below) are
crucial for obtaining maximum sensitivity.
In order to prevent acid-catalyzed lactonization of sialic
acids, glycans were dissolved in 0.2 M ammonium hydroxide
solution before being applied to the MALDI target plate
and air dried. The spot was rehydrated with water and a
mixture of THAP and ammonium citrate (1 mg THAP in
1 ml of a 1:1 mixture of acetonitrile and 20 mM ammonium citrate) was applied. Vacuum drying of the sample
spot was used to prevent large crystals from forming. The
sample was then allowed to absorb moisture from the
atmosphere to promote the formation of small crystals.
Figure 7 is representative of a MALDI-TOF MS spectrum
obtained in negative ion linear mode for the mono-sialylated N-linked glycan A1.
1932.69
100
831.06
945.43
896.98
1075.39
1299.76
1525.73
1653.00
1997.02
2072.01
2169.84
2313.50
2613.30
0
800
m/z
900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900
Figure 7. MALDI-TOF mass spectrum of A1 glycan obtained in negative ion linear mode using THAP as matrix and detected as the [M-H] ion.
sigma-aldrich.com
References
1. Stahl, B., Steup, M., Karas, M., and Hillenkamp, F., Anal. Biochem.,
63, 1463-1466 (1991).
2. Karas, M., Ehring, H., Nordhoff, E., Stahl, B., Strupat, K., Hillenkamp,
F., Grehl, M., and Krebs, B., Org. Mass. Spectrom., 28, 1476-1481 (1993).
3. Harvey, D.H., Rapid Commun. Mass Spectrom., 7, 614-619 (1993).
45

NMR Analysis of Glycans
Nuclear magnetic resonance (NMR) analysis is based on
the extent that a glycan distorts a magnetic field. This
technique is non-destructive and can provide full structural
information. Hence this is the first analysis technique that
can be used on a purified glycan. However, a major limitation of this technique is that a relatively large amount, in
the range 10-100 ng, of purified glycan is required. With
higher MHz spectrometers and use of nano-probes, an
increased sensitivity can be achieved. Although this is a
powerful method, the high capital cost of equipment and
the requirements of personnel trained in its use tend
to limit its application in the field of glycoprotein and
glycan analysis.
Electrophoresis Products
08658
8-Aminonapthalene 1,3,6-trisulfonic acid sodium

salt (ANTS)
06627
2-Aminoacridone (AMAC)
14,644-7
7-Amino-1,3-napthalenedisulfonic acid (ANDS)
15,615-9
Sodium cyanoborohydride
D 8779
Dimethylsulfoxide (DMSO)
A 4058
40% Acrylamide solution
M 7279
N,N-Methylenebisacrylamide (BIS)
T 4904
10x Tris Glycine buffer, pH 8.3
A 3678
Ammonium sulfate
T 8133
N,N,NN-Tetramethylenediamine (TEMED)
Electrophoresis of Glycans
Figure 8. Electrofluorogram of ANTS-labeled N-linked glycans.

Electrophoresis was carried out in 20% resolving polyacrylamide gel
for 1 hr at 250V. Lanes 1, 2, 3 and 4 represent Man-3, Man-7, Man-8
and NA4 glycans, respectively.
Glycan Analysis
and Labeling
Glycan Sequencing
Enzymatic analysis of oligosaccharides using highly specific
exoglycosidases, either sequentially or in a matrix array,
is a powerful technique to determine the sequence and
structure of glycan chains. Initially, the glycan pool can
be separated into individual oligosaccharides and each
of these purified glycans can be digested sequentially with
various linkage-specific exoglycosidase enzymes. Figure 9
shows some of the most commonly used exoglycosidase
enzymes used to determine the structures of N-linked
glycans. After appropriate incubation, the product of
digestion can be analyzed by normal phase HPLC or by
MALDI MS. On the HPLC system, structures are assigned to
each peak from the known specificity of the exoglycosidase
used in the digestion and the pre-determined incremental
gu values of individual monosaccharide residues.
Sequencing of oligosaccharides present in a glycan pool
can also be carried out simultaneously on aliquots of the
pool using enzyme arrays. Products of several digestions
are resolved on normal phase HPLC in separate runs and
the shift in the retention times of individual peaks, due
to the action of single enzyme, can be noted. As each
individual exoglycosidase removes additional monosaccharides from the glycan chain, the successive shift in the
retention times, and hence their corresponding gu values,
are obtained. By comparison with the experimentally
determined incremental gu values for the addition of
a particular sugar residue to the standard glycan core
structure, possible structure to each peak present in the
original glycan pool can be assigned. These structural
assignments can further be confirmed by tandem MSn
mass spectrometry.
sigma-aldrich.com
Separation of glycans by electrophoresis in polyacrylamide

gel has been widely used and different methods are
described in the literature for analysis of monosaccharides
and oligosaccharides. The most commonly used system
is the electrophoresis of fluorophore-labeled glycans in
highly cross-linked polyacrylamide gels and is termed as
Fluorophore-Assisted Carbohydrate Electrophoresis (FACE).
The glycans are usually labeled with a fluorescent tag,
mainly ANTS or AMAC and separated on 20-40% gels.
The extent of cross-linking means that extra precautions
should be taken to prevent heating and warping of the
gel during the run. After electrophresis, the band patterns
are visualized by illuminating the gel under UV light and
photographing the image. Although this technique is sensitive in the sub-picomolar range, the resolution between
the glycans can be poor due to the limitation on the size
of the gel. The following figure shows the mobility of
four individual N-linked glycans after labeling with ANTS.
This method can also be used to obtain profiles of glycans
released from glycoproteins and to elucidate the structure of individual glycans after exoglycosidase digestion,
based on the mobility shift of the parent glycan band.
46
Glycan Analysis
and Labeling
Figure 9. Some of the exoglycosidase enzymes commonly used to determine the structure of N-linked glycans.
sigma-aldrich.com
Exoglycosidases
A 6805
b-N-Acetylhexosaminidase
Recombinant, from Streptococcus pneumoniae, expressed in E. coli
F 8899
a-Fucosidase
From almond meal
F 5884
a-Fucosidase
From bovine kidney
G 4142
b-Galactosidase
From bovine testes
G 0413
b-Galactosidase
From Streptococcus pneumoniae
M 7257
a-Mannosidase
From Jack bean (Canavalia ensiformis)
N 3786
a-2-(3,6,8,9)-Neuraminidase
Proteomics grade, syn: a-sialidase, from Arthrobacter ureafaciens
N 7271
a-2,3-Neuraminidase
Recombinant, from Streptococcus pneumoniae, expressed in E. coli
47
M 8418
Man-3 Glycan
Syn: (Man)3(GlcNAc)2, Mannotriose-di-(N-acetyl-D-glucosamine), Oligomannose-3 glycan

70858-45-6, C34H58N2O26
M 2796
Man-3-F Glycan
Syn: M3F, Man3(Fuc)GlcNAc2, Mannotriose-[fucosyl-di-(N-acetyl-D-glucosamine)],

Oligomannose-3 glycan, core fucosylated
110387-51-4, C40H68N2O30
M 8912
Man-5 Glycan
Syn: (Man)5(GlcNAc)2, Mannopentaose-di-(N-acetyl-D-glucosamine), Oligomannose-5 glycan

66091-47-2, C46H78N2O36
Glycan and
Carbohydrate Standards
N-Linked High Mannose
sigma-aldrich.com
48

Man-5-Asn Glycan
Syn: (Man)5(GlcNAc)2Asn, Mannopentaose-di-(N-acetyl-D-glucosamine)-asparagine,

Oligomannose-5-Asn glycan
38784-68-8, C50H84N4O38
M 0420
Man-7 D1 Glycan
Syn: Man7(GlcNAc)2, Mannoheptaose-di-(N-acetyl-D-glucosamine) D1,

Oligomannose-7-D1 glycan
83178-05-06, C58H98N2O46
M 5918
Man-8 Glycan
Syn: Man8(GlcNAc)2, Mannooctaose-di-(N-acetyl-D-glucosamine) D1,D3,

Oligomannose-8 glycan
77036-51-2, C64H108N2O51
sigma-aldrich.com
Glycan and
M 4164
49

M 9037
Man-9 Glycan
Syn: (Man)9(GlcNAc)2, Mannononaose-di-(N-acetyl-D-glucosamine)

71246-55-4, C70H118N2O56
M 9919
HYBR glycan
Syn: GlcNAc(GlcNAc)Man5(GlcNAc)2, HYBR with bisecting GlcNAc, Mannopentaose-di(N-acetylglucosamine), N-acetylglucosaminyl-bisecting-N-acetylglucosaminyl

84182-21-8, C62H104N4O46
Glycan and
N-Linked Hybrid
N-Linked Complex
A1 Glycan
Syn: NeuNAc(Gal-GlcNAc)2Man3(GlcNAc)2, Mannotriose-di-(N-acetylglucosamine),

sialyl-bis(galactosyl-N-acetylglucosaminyl), Monosialylated, galactosylated,
biantennary glycan
145211-79-6, C73H121N5O54
sigma-aldrich.com
M 2425
50

N-Linked Complex
A1F Glycan
Syn: NeuNAc(Gal-GlcNAc)2Man3(GlcNAc)2Fuc, Mannotriose-(fucosyl-di-[N-acetylglucosamine]),

mono-sialyl-bis(galactosyl-N-acetylglucosaminyl), Monosialyl galactosyl biantennary glycan,
core fucosylated
571188-30-2, C79H131N5O8
M 5800
A2 Glycan
Mannotriose-di-(N-acetyl-D-glucosamine), bis([N-accetyl-D-neuraminyl]-galactosyl[N-acetyl-D-glucosaminyl]) ammonium salt, Disialyl galactosyl biantennary glycan

71496-55-4, C84H138N6O62
M 2800
A2F Glycan
Syn: (NeuNAc-Gal-GlcNAc)2Man2(Fuc)(GlcNAc)2, Mannotriose-(fucosyl-di[N-acetylglucosamine]), bis(sialyl-galactosyl-N-acetylglucosaminyl), Disialylated,

galactosylated, fucosylated biantennary glycan, Disialyl galactosyl biantennary glycan,
core fucosylated
108341-22-6, C90H148N6O66
sigma-aldrich.com
Glycan and
M 3800
51
NA2 Glycan
Syn: (Gal-GlcNAc)2Man3(GlcNAc)2, Mannotriose-di-(N-acetyl-D-glucosamine),

bis(galactosyl-[N-acetyl-D-glucosaminyl]), Asialo galactosyl biantennary glycan
71496-53-2, C62H104N4O46
M 2300
NA2B Glycan
Syn: (Gal-GlcNac)2(GlcNAc)Man3(GlcNAc)2, Mannotriose-di-(N-acetylglucosamine),

bis(galactosyl-N-acetylglucosaminyl)-bisecting N-acetylglucosaminyl, Asialo, galactosylated,
biantennary glycan, bisecting GlcNAc, Asialo galactosyl biantennary glycan, with
bisecting GlcNAc
84632-71-3, C70H117N5O51
M 9419
NA2F Glycan
Syn: (Gal)2(GlcNAc)2(Man)3(GlcNAc)2Fuc, Mannotriose-[fucosyl-di-(N-acetylglucosamine)],

bis(galactosyl-N-acetylglucosaminyl), Asialo, galactosylated, biantennary glycan, core
fucosylated, Asialo galactosyl biantennary glycan, core fucosylated
142561-43-1, C68H114N4O50
sigma-aldrich.com
M 5925
Glycan and
N-Linked Complex
52

N-Linked Complex
NGA2 Glycan
Syn: (GlcNAc)2Man3(GlcNAc)2, Mannotriose-di-(N-acetylglucosamine),

bis(N-acetylglucosaminyl), Asialo, agalacto, biantennary glycan, Asialo agalacto
biantennary glycan
84808-02-6, C50H84N4O36
M 1925
NGA2F Glycan
Syn: (GlcNAc)2Man3(Fuc)(GlcNAc)2, Mannotriose-[fucosyl-di-(N-acetylglucosamine)],

bis(N-acetylglucosaminyl), Asialo, agalacto, biantennary glycan, core fucosylated,
Asialo agalacto biantennary glycan, core fucosylated
84825-26-3, C56H94N4O40
M 1800
NGA2FB Glycan
Syn: (GlcNAc)Man3(Fuc)(GlcNAc)2, Mannotriose-[fucosyl-di-(N-acetylglucosamine)],

bis(N-acetylglucosaminyl)-bisecting N-acetylglucosaminyl), Asialo, agalacto, biantennary
glycan, bisecting GlcNAc, core fucosylated, Asialo agalacto biantennary glycan, core
fucosylated, with bisecting GlcNAc
79726-49-1, C64H107N5O45
sigma-aldrich.com
Glycan and
M 1675
53
M 2925
A3 Glycan
Syn: (NeuNAc-Gal-GlcNAc)3Man3(GlcNAc)2, Mannotriose-di-(N-acetyl-D-glucosamine),

tris(sialyl-galactosyl-N-acetyl-D-glucosaminyl, Trisialylated, galactosylated, triantennary glycan,
Trisialyl galactosyl triantennary glycan
145164-24-5, C109H178N8O80
M 8793
NA3 Glycan
Syn: (Gal-GlcNAc)3-Man3-GlcNAc2, Mannotriose-di-(N-acetyl-D-glucosamine),

tris(galactosyl-N-acetyl-D-glucosaminyl, Asialo, galactosylated, triantennary glycan,
Asialo galactosyl triantennary glycan
82867-73-0, C76H127N5O56
Glycan and
N-Linked Complex
sigma-aldrich.com
54

N-Linked Complex
NGA3 Glycan
Syn: (GlcNAc)3Man3GlcNAc2, Mannotriose-di-(N-acetyl-D-glucosamine),

tris(N-acetyl-D-glucosaminyl), Asialo, agalacto, triantennary glycan,
Asialo agalacto triantennary glycan
110387-63-8, C58H97N5O41
M 8918
NA4 Glycan
Syn: [Gal-GlcNAc]4-Man3-GlcNAc2, Mannotriose-di-(N-acetylglucosamine), tetrakis

(galactosyl-N-acetyl-D-glucosaminyl), Asialo, tetraantennary N-linked glycan Asialo
galactosyl tetraantennary glycan
82867-74-1, C90H150N6O66
Glycan and
M 2175
sigma-aldrich.com
55
N-Asn
Syn: 2-Acetamido-1-b-(L-aspartamido)-1,2-dideoxy-D-glucose, b-D-GlcNAc-1N-Asn,

2-Acetamido-1-N-(b-L-aspartyl)-2-deoxy-b-D-glucopyranosylamine
2776-93-4, C12H21N3O8
A 3432
N2-Asn
Syn: N,N-Diacetyl-1-(N-b-L-aspartyl)-b-chitobiose, b-D-GlcNAc-(14)-b-D-GlcNAc-1-N-Asn,

2-Acetamido-4-O-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-1-N-(b-L-aspartyl)-2-deoxy-bD-glucopyranosylamine
29625-73-8, C20H34N4O13
A 2292
MN Glycan
Syn: b-D-Man-(14)-D-GlcNAc, 2-Acetamido-2-deoxy-4-O-(b-D-mannopyranosyl)-Dglucopyranose

186765-90-2, C14H25NO11
A 6587
NM Glycan
Syn: b-D-GlcNAc-(12)-D-Man, 2-O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-D-mannose

34621-73-3, C14H25NO11
A 5065
3a-Fucosyl-N-acetylglucosamine
Syn: a-L-Fuc-(13)-D-GlcNAc, 2-Acetamido-2-deoxy-3-O-a-L-fucopyranosyl-D-glucopyranose

24876-86-6, C14H25NO10
A 9566
4a-Fucosyl-N-acetylglucosamine
Syn: a-L-Fuc-(14)-D-GlcNAc, 2-Acetamido-2-deoxy-4-O-a-L-fucopyranosyl-D-glucopyranose

76211-71-7, C14H25NO10
M 1050
2a-Mannobiose
Syn: a-D-Man-(12)-D-Man, 2-O-a-D-Mannopyranosyl-D-mannopyranose

15548-39-7, C12H22O11
sigma-aldrich.com
A 6681
Glycan and
N-Linked Fragments
56
Glycan and
N-Linked Fragments
M 6155
4a-Mannobiose
Syn: a-D-Man(14)-D-Man, 4-O-a-D-Mannopyranosyl-D-mannopyranose

35438-40-5, C12H22O11
M 7788
6a-Mannobiose
Syn: a-D-Man-(16)-D-Man, 6-O-a-D-Mannopyranosyl-D-mannopyranose

6614-35-3, C12H22O11
D 5422
3a,6a-Mannotriose
Syn: a-D-Man-(13)-[a-D-Man-(16)]-D-Man, 3,6-Di-O-(a-D-mannopyranosyl)-Dmannopyranose

121123-33-9, C18H32O16
M 0925
3a,6a-Mannopentaose
Syn: a-Man-(13)(a-Man-[16])-a-Man-(16)(a-Man-[13])-Man
112828-69-0, C30H52O26
sigma-aldrich.com
57
N-Acetyllactosamine
Syn: N-Acetyl-4-O-(b-D-galactopyranosyl)-D-glucosamine, b-D-Gal-(14)-D-GlcNAc,

2-Acetamido-2-deoxy-4-O-b-D-galactopyranosyl-D-glucopyranose, N-acetyl-D-lactosamine
32181-59-2, C14H25NO11
A 6919
6b-GlcNAc-D-galactose
Syn: b-D-GlcNAc-(16)-D-Gal, 6-O-(2-Acetamido-2-deoxy-b-D-glucopyranosyl)-Dgalactopyranose, b6-GlcNAc-(16)-D-galactose

20212-77-5, C14H25NO11
A 8297
6b-GlcNAc-lactose
Syn: b-D-GlcNAc-(16)-b-D-Gal-(14)-D-Glc, 6-N-Acetylglucosaminyllactose,

4-O-(6-O-[2-Acetamido-2-deoxy-b-D-glucopyranosyl]-b-D-galactopyranosyl)-D-glucopyranose
68665-69-0, C20H35NO16
F 0393
2-Fucosyllactose
Syn: a-L-Fuc-(12)-b-D-Gal-(14)-D-Glc, 2-FL, 2-fucosyl-D-lactose

41263-94-9, C18H32O15
F 9641
3-Fucosyllactose
Syn: a-L-Fuc-(13)-[b-D-Gal-(14)]-D-Glc, 3-FL

41312-47-4, C18H32O15
L 6770
Lacto-N-tetraose
Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNT

14116-68-8, C26H45NO21
L 8526
Lacto-N-neo-tetraose
Syn: b-D-Gal-(14)-b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNnT

13007-32-4, C26H45NO21
sigma-aldrich.com
A 7791
Glycan and
O-Linked Neutral Glycans
58
sigma-aldrich.com
Glycan and

L 8651
LDFT Glycan
Syn: 3,2-Difucosyllactose, a-L-Fuc-(12)-b-D-Gal-(14)-[a-L-Fuc-(13)]-D-Glc,

Lactodifucotetraose
20768-11-0, C24H42O19
L 5908
Lacto-N-fucopentaose I
Syn: a-L-Fuc (12)-b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNFP I

7578-25-8, C32H55NO25
L 6401
Lacto-N-fucopentaose II
Syn: Lea-Lactose, Lewis-a pentasaccharide, b-D-Gal-(13)-[a-L-Fuc-(14)]-b-D-GlcNAc(13)-b-D-Gal-(14)-D-Glc, LNFP II

21973-23-9, C32H55NO25
L 7777
Lacto-N-fucopentaose III
Syn: Lex-lactose, Lewis-X pentasaccharide, b-D-Gal-(14)-[a-L-Fuc-(13)]-b-D-GlcNAc(13)-b-D-Gal-(14)-D-Glc, LNFP III

25541-09-7, C32H55NO25
L 7902
Lacto-N-fucopentaose V
Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-[a-L-Fuc-(13)]-D-Glc, LNFP V

60254-64-0, C32H55NO25
59
Lacto-N-difucohexaose I
Syn: Leb-lactose, Lewis-b hexasaccharide, a-L-Fuc-(12)-b-D-Gal-(13)[a-L-Fuc-(14)]b-D-GlcNAc-(13)-b-D-Gal-(14)-D-Glc, LNDFH I

16789-38-1, C38H65NO29
L 6645
Lacto-N-difucohexaose II
Syn: b-D-Gal-(13)-[a-L-Fuc-(14)]-b-D-GlcNAc-(13)-b-D-Gal-(14)-[a-L-Fuc-(13)]D-Glc, LNDFH II

62258-12-2, C38H65NO29
L 6654
Lacto-N-hexaose
Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-(b-D-Gal-[14]-b-D-GlcNAc-[16])-b-D-Gal(14)-D-Glc, LNH

64003-51-6, C40H68N2O31
L 8283
para-Lacto-N-hexaose
Syn: b-D-Gal-(13)-b-D-GlcNAc-(13)-b-D-Gal-(14)-b-D-GlcNAc-(13)-b-D-Gal-(14)D-Glc, pLNH

64331-48-2, C40H68N2O31
F 5171
Fucosyl-para-lacto-N-hexaose IV
Syn: b-Gal-(13)-b-GlcNAc-(13)-b-Gal-(1-4)(a-Fuc-[13])-b-GlcNAc-(13)-b-Gal-(14)Glc, FpLNH IV

115236-58-3, C46H78N2O35
sigma-aldrich.com
L 7033
Glycan and
60
sigma-aldrich.com
Glycan and
O-Linked Sialylated Glycans

A 1314
3-Sialyl-3-fucosyllactose
Syn: a-NeuNAc-(23)-b-D-Gal-(14)-(a-Fuc-[13])-Glc, SFL,

3-N-acetylneuraminyl-3-fucosyllactose
122560-33-2, C29H48NNaO23
A 4564
LS-Tetrasaccharide a
Syn: LST a, a-NeuNAc-(23)-b-Gal-(13)-b-GlcNAc-(13]-b-Gal-(14)-Glc,

N-Acetylneuraminyllacto-N-tetraose a
64003-53-8, C37H62N2O29
A 4689
LS-Tetrasaccharide b
Syn: LST b, a-NeuNAc-(26)-(b-D-Gal-[13])-b-D-GlcNAc-(13)-b-D-Gal-(14)-Glc,

N-Acetylneuraminyllacto-N-tetraose b
64003-54-9, C37H62N2O29
A 4814
LS-Tetrasaccharide c
Syn: LST c, a-NeuNAc-(26)-b-Gal-(14)-b-GlcNAc-(13)-b-Gal-(14)-Glc,

N-Acetylneuraminyllacto-N-neo-tetraose c
64003-55-0, C37H61N2NaO29
61
D 9045
Disialyllacto-N-tetraose
Syn: DSLNT, a-NeuNAc-(23)-b-Gal-(13)-[a-NeuNAc-(26)]-b-GlcNAc-(13)-b-Gal-(14)-Glc,

a-Neu5Ac-(23)-b-Gal-(13)-[a-Neu5Ac-(26)]-b-GlcNAc-(13)-b-Gal-(14)-Glc
61278-38-4, C48H77N3Na2O37
A 8681
3-Sialyllactose
from bovine colostrum
3-Sialyllactose
from human milk
Syn: NANA-Lactose, 3-N-Acetylneuraminyllactose, a-NeuNAc-(23)-b-D-Gal-(14)-D-Glc,

N-Acetylneuraminyllactose, 3-SL, a-Neu5Ac-(23)-b-D-Gal-(14)-D-Glc
35890-38-1, C23H39NO19
6-Sialyllactose
from human milk
6-Sialyllactose
Syn: a-NeuNAc-(26)-b-D-Gal-(14)-D-Glc, 6-N-Acetylneuraminyllactose, 6-SL,

a-Neu5Ac-(26)-b-D-Gal-(14)-D-Glc
74609-39-5, C23H38NNaO19
A 9079
A 9204
A 8556
Glycan and
sigma-aldrich.com
62
Glycan and

A 6936
3-Sialyl-N-acetyllactosamine
Syn: a-NeuNAc-(23)-b-D-Gal-(14)-D-GlcNAc, 3-N-Acetylneuraminyl-N-acetyllactosamine,

3-SLN
81693-22-3, C25H42N2O19
A 8431
6-Sialyl-N-acetyllactosamine
Syn: a-NeuNAc-(26)-b-D-Gal-(14)-D-GlcNAc, 6-N-Acetylneuraminyl-N-acetyllactosamine,

6-SLN, a-Neu5Ac-(26)-b-D-Gal-(14)-D-GlcNAc
136098-09-4, C25H41N2NaO19
A 0828
Sialyllactose
from human milk
Syn: N-Acetylneuraminyllactose, a-NeuNAc-(26)- and -(23)-b-D-Gal-(14)-D-Glc,

Neuramin-lactose, a-Neu5Ac-(26)- and -(26)-b-D-Gal-(14)-D-Glc
C23H38NNaO19
A 3307
Sialyllactose
Syn: N-Acetylneuraminyl-lactose, a-NeuNAc-(23)- and -(26)-b-D-Gal-(14)-D-Glc,

Neuramin-lactose, a-Neu5Ac-(23)- and -(26)-b-D-Gal-(14)-D-Glc
C23H39NO19
sigma-aldrich.com
63
4a-Galactobiose
Syn: a-D-Gal-(14)-D-Gal, 4-O-a-D-Galactopyranosyl-D-galactopyranose

80446-85-1, C12H22O11
G 9662
4b-Galactobiose
Syn: b-D-Gal-(14)-D-Gal, 4-O-b-D-Galactopyranosyl-D-galactopyranose

2152-98-9, C12H22O11
A 0167
Galacto-N-biose
Syn: b-D-Gal-(13)-D-GalNAc, 2-Acetamido-2-deoxy-3-O-b-D-galactopyranosylD-galactopyranose, T-Antigen

20972-29-6, C14H25NO11
A 9150
Lacto-N-biose
Syn: b-D-Gal-(13)-D-GlcNAc, 2-Acetamido-2-deoxy-3-O-(b-D-galactopyranosyl)D-glucopyranose

50787-09-2, C14H25NO11
F 7012
H-Disaccharide
Syn: a-L-Fuc-(12)-D-Gal, 2-O-a-L-Fucosyl-D-galactose

24656-24-4, C12H22O10
G 8394
4b-Galactosyllactose
Syn: b-D-Gal-(14)-b-D-Gal-(14)-D-Glc, 4-O-(4-O-b-D-Galactopyranosylb-D-galactopyranosyl)-D-glucopyranose

118396-93-3, C18H32O16
G 9287
Globotriose
Syn: a-D-Gal-(14)-b-D-Gal-(14)-D-Glc, 4-O-(4-O-a-D-Galactopyranosylb-D-galactopyranosyl)-D-glucopyranose

66580-68-5, C18H32O16
sigma-aldrich.com
G 8281
Glycan and
Blood Group Antigens
64
sigma-aldrich.com
Glycan and

A 7911
A-Trisaccharide
Syn: Blood group A trisaccharide, a-L-Fuc-(12)-[a-D-GalNAc-(13)]-D-Gal

49777-13-1, C20H35NO15
B 1422
B-Trisaccharide
Syn: Blood group B trisaccharide, a-L-Fuc-(12)(a-D-Gal-[13])-D-Gal

49777-14-2, C18H32O15
F 7297
H-Trisaccharide
Syn: Blood Group H trisaccharide, a-Fuc-(12)-b-Gal-(14)-GlcNAc,

2-Fucosyl-N-acetyllactosamine
C2H35NO16
A 8956
A-Tetrasaccharide
Syn: a-D-GalNAc-(13)[a-L-Fuc-(12)]-b-D-Gal-(14)-D-Glc
59957-92-5, C26H45NO20
A 1955
A-Pentasaccharide
Syn: a-D-GalNAc-(13)-[a-L-Fuc-(12)]-b-D-Gal-(14)-[a-L-Fuc-(13)]-D-Glc
50624-46-9, C32H55NO24
65
B 3791
B-Pentasaccharide
Syn: a-Fuc-(12)[a-Gal-(13)]-b-Gal-(14)[a-Fuc-(13)]-Glc
72468-43-0, C30H52O24
A 7560
iso-A-Pentasaccharide
Syn: a-GalNAc-(13)[a-Fuc-(12)]-b-Gal-(13)-[a-Fuc-(14)]-Glc,
A-Leb-Pentasaccharide
128464-25-5, C32H55NO24
B 0799
iso-B-Pentasaccharide
Syn: a-Fuc-(12)[a-Gal-(13)]-b-Gal-(13)[a-Fuc-(14)]-Glc,
b-Leb-Pentasaccharide
128464-26-6, C30H52O24
Glycan and
L 7276
Lewis-a trisaccharide
Syn: a-L-Fuc-(14)-[b-D-Gal-(13)]-D-GlcNAc, Lea trisaccharide

56570-03-7, C20H35NO15
L 5152
Lewis-X trisaccharide
Syn: a-L-Fuc-(13)-[b-D-Gal-(14)]-D-GlcNAc, Lex trisaccharide

71208-06-5, C20H35NO15
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Lewis and Cell Adhesion Glycans
66
sigma-aldrich.com
Glycan and
Lewis and Cell Adhesion Glycans

L 7659
Lewis-b tetrasaccharide
Syn: a-Fuc(12)-b-Gal-(13)-[a-Fuc-(14)]-GlcNAc, Leb tetrasaccharide

C38H67NO29
L 7784
Lewis-Y tetrasaccharide
Syn: a-Fuc-(12)-b-Gal-(14)-[a-Fuc-(13)]-GlcNAc, LeY tetrasaccharide

C38H67NO29
L 7401
Lewis-Y hexasaccharide
Syn: a-Fuc-(12)-b-Gal-(14)(a-Fuc-[13])-b-GlcNAc-(13)-b-Gal-(14)-Glc, Ley-lactose,

Lacto-N-neo-difucohexaose I, LeY hexasaccharide
62469-99-2, C38H65NO29
S 2279
3-Sialyl-Lewis-a tetrasaccharide
Syn: a-NeuNAc-(23)-b-D-Gal-(13)-[a-L-Fuc-(14)]-D-GlcNAc, 3-SLea

92448-22-1, C31H52N2O32
S 1782
3-Sialyl-Lewis-X tetrasaccharide
Syn: a-NeuNAc-(23)-b-D-Gal-(14)[a-L-Fuc-(13)]-D-GlcNAc, 3-SLeX

98603-84-0, C31H52N2O23
67

Gal a(1-3) Gal Antigens
G 7522
3a-Galactobiose
Syn: a-D-Gal-(13)-D-Gal, 3-O-a-D-Galactopyranosyl-D-galactose

13168-24-6, C12H22O11
G 9787
3a,4b-Galactotriose
Syn: a-D-Gal-(13)-b-D-Gal-(14)-D-Gal, 4-O-(3-O-a-D-Galactopyranosylb-D-galactopyranosyl)-D-galactopyranose

56038-36-9, C18H32O16
G 9912
3a,4b,3a-Galactotetraose
Syn: a-D-Gal-(13)-b-D-Gal-(14)-b-D-Gal-(13)-D-Gal, 3-O-(4-O-[3-O-a-D-Galactopyranosylb-D-galactopyranosyl]-a-D-galactopyranosyl)-D-galactopyranose

56038-38-1, C24H42O21
M 5107
IgM, Lambda
from murine myeloma
Clone MOPC 104E, Purified immunoglobulin, Buffered aqueous solution
M 2521
IgM, Lambda
from murine myeloma
Clone MOPC 104E, Ascites fluid, Lyophilized powder
G 7528
D-Glucose
Syn: Grape sugar, Dextrose, Corn sugar

50-99-7, SigmaUltra 99.5% (GC), C6H12O6
G 6404
D-Galactose
59-23-4, SigmaUltra minimum 99%, C6H12O6
M 8296
D-Mannose
Syn: D-Mannopyranose
3458-28-4, minimum 99% SigmaUltra, C6H12O6
F 2252
L-Fucose
Syn: 6-Deoxy-L-galactose
2438-80-4, minimum 99%, C6H12O5
X 3877
D-Xylose
58-86-6, SigmaUltra 99%, C5H10O5
A 8625
N-Acetyl-D-glucosamine
Syn: 2-Acetamido-2-deoxy-D-glucose, D-GlcNAc

7512-17-6, C8H15NO6
A 2795
N-Acetyl-D-galactosamine
Syn: D-GalNAc, N-Acetylchondrosamine, 2-Acetamido-2-deoxy-D-galactose

1811-31-0, C8H15NO6
A 0812
N-Acetylneuraminic acid
Syn: NeuNAc, Sialic acid, Lactaminic acid, NANA, 5-Acetamido-3,5-dideoxy-D-glyceroD-galactononulosonic acid, NAN
131-48-6, Type IV-S, C11H19NO9
G 2755
N-Glycolylneuraminic acid
Syn: NeuNGl
1113-83-3, C11H19NO10
G 8645
D-Glucuronic acid sodium salt
Syn: Sodium D-glucuronate

14984-34-0, C6H9NaO7 H2O
Glycan and
Gal a(1-3) Gal Antigens
Monosaccharides
sigma-aldrich.com
68
Glycobiology Product Directory

Additional Information Sources
Visit the Sigma-Aldrich Glycoprotein Analysis home page at sigma-aldrich.com/glyco
This new and growing web page contains:
Hundreds of enzymes, glycoproteins,
substrates, and reagents for glycoprotein
analysis
Links to additional enzymes, inhibitors,
antibodies, and lectins as well as related
products available through the
Enzyme Explorer
Product information, MSDS, and
Certificates of Analysis
For direct link to the most up-to-date
new product information visit
Glycobiology
Product Directory
Sigma-Aldrich Enzyme Explorer

sigma-aldrich.com/enzymeexplorer
Tap into the most comprehensive line of enzymes, substrates, and inhibitors in the industry. The Sigma Enzyme
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finding the products and information you need.
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The Enzyme Class Index helps you find enzymes by their
EC number or by the type of reaction they catalyze.
The Enzyme Inhibitor Index contains over 600 inhibitor
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69
Enzymes
Complete List of Glycolytic Enzymes
A 9763
a-N-Acetylgalactosaminidase
From chicken liver, lyophilized powder, 5-20 units/mg protein
A 6680
N-Acetylneuraminic acid Aldolase
Expressed in E. coli
A 6306
Agarase
Pseudomonas atlantica, lyophilized powder
A 4551
a-Amylase
From Bacillus licheniformis, 500-1,500 units/mg, lyophilized powder
A 6814
a-Amylase
From Bacillus sp., minimum 400 units/mg
A 6380
a-Amylase
From Bacillus sp., type II-A, lyophilized powder
A 6211
a-Amylase
From Aspergillus oryzae, 150-250 units/mg
A 2771
a-Amylase
From barley malt, type VIII-A
A 0521
a-Amylase
From human saliva, type IX-A, lyophilized powder
A 1031
a-Amylase
From human saliva, type XIII-A, lyophilized powder
A 4268
a-Amylase
From porcine pancreas, type I-A, PMSF treated saline suspension
A 6255
a-Amylase
From porcine pancreas, type I-A, saline suspension
A 2643
a-Amylase
From porcine pancreas, type VII-A, ammonium sulfate suspension
A 3403
a-Amylase
From Bacillus licheniformis, type XII-A, saline solution
A 3306
a-Amylase
Heat-stable, solution
A 7130
b-Amylase
From barley, type II-B
A 7005
b-Amylase
From sweet potato, type I-B, ammonium sulfate suspension
I 2758
Isoamylase
From Pseudomonas amyloderamosa, ammonium sulfate suspension,

minimum 2,000,000 units/mg
Amylase
A 7420
Amyloglucosidase
From Aspergillus niger, lyophilized powder
A 7095
Amyloglucosidase
From Aspergillus niger, minimum 300 units/ml
A 2330
Amyloglucosidase
From Candida tsukubaensis, ammonium sulfate suspension
A 7255
Amyloglucosidase
From Rhizopus sp., minimum 5,000 units/g
A 3042
Amyloglucosidase
From Aspergillus niger, 5,000-8,000 units/ml, aqueous glucose solution
A 3514
Amyloglucosidase
From Aspergillus niger, ammonium sulfate suspension, 40-80 units/mg
C 1184
Cellulase
From Aspergillus niger, powder, minimum 0.3 units/mg
C 2605
Cellulase
From Aspergillus sp., minimum 1,000 units/g
C 2730
Cellulase
From Trichoderma reesei, ATCC 26921, liquid, minimum 700 units/g
C 9422
Cellulase
From Trichoderma viride, crude powder, 3-10 units/mg
C 0898
Cellulase
From Trichoderma viride, lyophilized powder
C 7809
Chitinase
From Serratia marcescens, lyophilized powder, 400-1,200 units/g
C 1650
Chitinase
From Serratia marcescens, lyophilized powder, minimum 10 units/g
C 6137
Chitinase
From Streptomyces griseus, lyophilized powder, 200-600 units/g
C 1525
Chitinase
From Streptomyces griseus, lyophilized powder, 500-2,000 units/g
C 9830
Chitosanase
From Streptomyces griseus, lyophilized powder, >50 units/mg
C 0794
Chitosanase
From Streptomyces sp., buffered aqueous glycerol solution, minimum 15 units/mg
Glycobiology
Product Directory
Amyloglucosidase
Cellulase
Chitinase
Chondroitinase
C 2905
Chondroitinase ABC
From Proteus vulgaris, lyophilized powder, 0.3-3 units/mg
C 3667
Chondroitinase ABC
From Proteus vulgaris, lyophilized powder, 50-250 units/mg
sigma-aldrich.com
Chitosanase
70
Enzymes
Chondroitinase (cont)
C 2262
Chondroitinase AC
From Arthrobacter aurescens, vial of minimum 5 units
C 2780
Chondroitinase AC
From Flavobacterium heparinum, lyophilized powder, 0.5-1.5 units/mg
C 0954
Chondroitinase C
From Flavobacterium heparinum, lyophilized powder, minimum 200 units/mg
C 8058
Chondroitinase B
From Flavobacterium heparinum, lyophilized powder, 100-300 units/mg
Chondrosulfatase
C 2655
Chondro-4-sulfatase
From Proteus vulgaris, lyophilized powder, approx. 10 units/mg
C 3030
Chondro-6-sulfatase
From Proteus vulgaris, lyophilized powder, 3-10 units/mg
C 8274
Cytohelicase
From Helix pomatia, lyophilized powder
D 9909
Dextran sucrase
From Leuconostoc mesenteroides, lyophilized powder, minimum 100 units/mg
D 4668
Dextranase
From Penicillium sp., lyophilized powder, 100-200 units/mg
D 5884
Dextranase
D 8144
Dextranase
D 9515
Driselase
From Basidiomycetes sp., crude powder containing laminarinase, xylanase and cellulase
Dextranase
Glycobiology
Product Directory
Endoglycosidase
E 9762
Endoglycosidase F1
From Chryseobacterium (Flavobacterium) meningosepticum, 16 units/mg
E 0639
Endoglycosidase F2
E 2264
Endoglycosidase F3
E 6878
Endoglycosidase H
From Streptomyces griseus, lyophilized powder
A 0810
Endoglycosidase H
From Streptomyces plicatus, recombinant, expressed in E. coli, buffered aqueous solution
E 7642
Endoglycosidase H
From Streptomyces plicatus, recombinant, expressed in E. coli, buffered aqueous solution
F 1924
a1-(2,3,4)-Fucosidase
From Xanthomonas sp., buffered aqueous solution
F 9272
a1,2-Fucosidase
Buffered aqueous solution
F 3023
a1-(3,4)-Fucosidase
Buffered aqueous solution
F 6272
a1,6-Fucosidase
Recombinant, expressed in E. coli, buffered aqueous solution
F 8899
a-Fucosidase
From almonds, lyophilized powder
F 6151
a-L-Fucosidase
From human placenta, ammonium sulfate suspension, minimum 2 units/mg
F 5884
a-L-Fucosidase
From bovine kidney, ammonium sulfate suspension, minimum 2 units/mg
Fucosidase
sigma-aldrich.com
Galactosidase
G 7163
a-Galactosidase
Recombinant, expressed in E. coli, buffered aqueous solution, positionally specific solution
G 4408
a-Galactosidase
G 8507
a-Galactosidase
G 1288
b1-(3,4,6)-Galactosidase
Recombinant, expressed in E. coli, buffered aqueous solution, positionally specific
G 0288
b1-(3,6)-Galactosidase
G 0413
b1,4-Galactosidase
From Streptococcus pneumoniae, recombinant, expressed in E. coli, buffered aqueous

solution, positionally specific
G 0914
b1,6-Galactosidase
G 3153
b-Galactosidase
Recombinant, expressed in E. coli, overproducing strain, lyophilized powder,

minimum 500 units/mg
G 5635
b-Galactosidase
Expressed in E. coli, grade VIII, lyophilized powder, 600-1,200 units/mg
G 5160
b-Galactosidase
From Aspergillus oryzae, minimum 8 units/mg solid
G 4142
b-Galactosidase
From bovine testes, ammonium sulfate suspension, 1-3 units/mg
G 4155
b-Galactosidase
Expressed in E. coli, aqueous glycerol suspension, 600-1,200 units/mg
71
Enzymes
Galactosidase (cont)
G 6008
b-Galactosidase
Expressed in E. coli, grade VI, lyophilized powder, 250-600 units/mg
G 2513
b-Galactosidase
Expressed in E. coli, grade X, ammonium sulfate suspension, minimum 250 units/mg
G 1875
b-Galactosidase
From bovine liver, grade III, lyophilized powder, approx. 0.15 units/mg
G 3782
b-Galactosidase
From Saccharomyces fragilis, aqueous glycerol solution, 4-12 units/mg
G 5507
Galactosyltransferase
From bovine milk, lyophilized powder, 3-15 units/mg
G 2133
Glucose Oxidase
From Aspergillus niger
G 8162
b-Glucuronidase
Expressed in E. coli, aqueous glycerol solution, 20,000,000-60,000,000 units/g
G 7396
b-Glucuronidase
Expressed in E. coli, type IX-A, lyophilized powder, 1,000,000-5,000,000 units/g
G 7646
b-Glucuronidase
Expressed in E. coli, type VII-A, lyophilized powder, 5,000,000-20,000,000 units/g
G 7771
b-Glucuronidase
Expressed in E. coli, type VIII-A, aqueous glycerol solution, 5,000,000-20,000,000 units/g
G 7896
b-Glucuronidase
Expressed in E. coli, type X-A, lyophilized powder, 20,000,000-60,000,000 units/g
G 5897
b-Glucuronidase
Expressed in E. coli, vial of minimum 1,000 units
G 8271
b-Glucuronidase
Expressed in E. coli, vial of minimum 1,000 units
G 0251
b-Glucuronidase
From bovine liver, type B-1
G 0501
b-Glucuronidase
From bovine liver, type B-10, approx. 10,000 units/mg
G 0376
b-Glucuronidase
From bovine liver, type B-3, approx. 3,000 units/mg
G 0751
b-Glucuronidase
From Helix pomatia, type H-1, minimum 300,000 units/g
G 0876
b-Glucuronidase
From Helix pomatia, type H-2, minimum 100,000 units/ml
G 8885
b-Glucuronidase
From Helix pomatia, type H-3, approx. 100,000 units/ml
G 0762
b-Glucuronidase
From Helix pomatia, type H-3AF, crude solution, minimum 60,000 units/ml
G 1512
b-Glucuronidase
From Helix pomatia, type H-5, minimum 400,000 units/g
G 7017
b-Glucuronidase
From Helix pomatia, type HP-2, minimum 100,000 units/ml
G 7770
b-Glucuronidase
From Helix pomatia, type HP-2S, approx. 100,000 units/ml
G 8132
b-Glucuronidase
From Patella vulgata (keyhole limpet), type L-II, lyophilized powder, 1,000,000-3,000,000 units/g
G 4259
b-Glucuronidase
From Helix aspersa (garden snail), 250,000-500,000 units/g
Glycobiology
Product Directory
Glucuronidase
Glucosaminidase
A 7708
b-N-Acetylglucosaminidase
From Aspergillus oryzae
A 2415
From bovine kidney, ammonium sulfate suspension
A 6152
From human placenta, ammonium sulfate suspension
A 6805
From Streptococcus pneumoniae, recombinant, expressed in E. coli, buffered aqueous solution
A 2264
From Canavalia ensiformis (jack bean), ammonium sulfate suspension
A 3189
From Aspergillus niger, ammonium sulfate suspension
A 3391
b-N-Acetylglucosaminidase A
From bovine epididymis, ammonium sulfate suspension,

0.25-1.5 units/mg protein (modified Warburg-Christian)
A 7640
b-N-Acetylglucosaminidase B
From bovine epididymis, ammonium sulfate suspension, 5-10 units/mg
G 3651
a-Glucosidase
From Bacillus stearothermophilus, lyophilized powder, minimum 50 units/mg
G 0660
a-Glucosidase
From Saccharomyces cerevisiae, recombinant, expressed in unspecified host,

lyophilized powder, minimum 125 units/mg
G 5003
a-Glucosidase
From Saccharomyces cerevisiae, type I, minimum 10 units/mg
G 6906
b-Glucosidase
From Caldocellum saccharolyticum, recombinant, expressed in E. coli, lyophilized powder,

minimum 300 units/g
G 4511
b-Glucosidase
From almonds, lyophilized powder, 20-40 units/mg
G 0395
b-Glucosidase
From almonds, lyophilized powder, minimum 2 units/mg
sigma-aldrich.com
Glucosidase
72
Enzymes
G 9028
d4,5-Glycuronidase
From Flavobacterium heparinum, aqueous solution, 20 units/mg
G 1163
O-Glycosidase
From Streptococcus pneumoniae, recombinant, expressed in E. coli,

buffered aqueous solution
H 2125
Hemicellulase
From Aspergillus niger, 0.01-0.1 unit/mg solid
H 2519
Heparinase I
From Flavobacterium heparinum, lyophilized powder,

stabilized with approx. 25% bovine serum albumin, 200-600 units/mg
H 6512
Heparinase II
From Flavobacterium heparinum, lyophilized powder, 100-300 units/mg
H 8891
Heparinase III
From Flavobacterium heparinum, lyophilized powder, 200-600 units/mg solid
Heparinase
Hesperidinase
H 8137
Hesperidinase
From Aspergillus niger, lyophilized powder, minimum 1 unit/g
H 8510
Hesperidinase
From Penicillium sp., lyophilized powder, minimum 10 units/g
Glycobiology
Product Directory
Hyaluronidase
H 3506
Hyaluronidase
From bovine testes, type I-S, lyophilized powder
H 3884
Hyaluronidase
From bovine testes, type IV-S, essentially salt-free, lyophilized powder
H 3757
Hyaluronidase
From bovine testes, type VIII, lyophilized powder, approx. 300 units/mg
H 3631
Hyaluronidase
From bovine testes, type VI-S, lyophilized powder, 3,000-15,000 units/mg
H 2126
Hyaluronidase
From sheep testes, type II, lyophilized powder
H 2251
Hyaluronidase
From sheep testes, type III, lyophilized powder
H 6254
Hyaluronidase
From sheep testes, type V, lyophilized powder, minimum 1,500 units/mg
H 1136
Hyaluronidase
From Streptomyces hyalurolyticus, lyophilized powder
I 9274
Invertase
From bakers yeast (S. cerevisiae), approx. 300 units/mg
I 9253
Invertase
From bakers yeast (S. cerevisiae), grade V, minimum 30 units/mg
I 4504
Invertase
From bakers yeast (S. cerevisiae), grade VII, minimum 400 units/mg
I 4753
Invertase
From Candida utilis, grade X, 300-500 units/mg
L 9903
Lacto-N-biosidase
From Streptococcus sp.
Invertase
Laminarinase
L 9259
Laminarinase
L 5272
Laminarinase
From Trichoderma sp., 100-400 units/g
L 7773
Lysozyme
From chicken egg white, aseptically filled
L 7001
Lysozyme
From chicken egg white, grade III, approx. 50,000 units/mg
L 6876
Lysozyme
From chicken egg white, lyophilized powder, approx. 50,000 units/mg
L 6394
Lysozyme
From human milk, lyophilized powder, minimum 100,000 units/mg
L 8402
Lysozyme
From human neutrophils, minimum 95% (SDS-PAGE), lyophilized powder,

minimum 100,000 units/mg
L 1129
Lysozyme Agarose
From chicken egg white, lyophilized powder, 5,000-10,000 units/g
M 1266
Lysozyme Agarose
From chicken egg white, lyophilized powder, 5,000-10,000 units/g
L 2879
Lysozyme chloride form
From chicken egg white, grade VI, not dialyzed or lyophilized, approx. 60,000 units/mg
sigma-aldrich.com
Lysozyme
Mannosidase
M 7257
a-Mannosidase
M 2658
a-Mannosidase
Buffered aqueous solution, expressed in E. coli, Recombinant
M 9400
b1,4-Mannosidase, b-Mannosidase
From Helix pomatia, From snail acetone powder, ammonium sulfate suspension, 5-30 units/ml
From Canavalia ensiformis (jack bean), ammonium sulfate suspension, approx. 20 units/mg
73
Enzymes
N 1385
Naringinase
From Penicillium decumbens, 300 units/g
N 7271
a2,3-Neuraminidase
From Streptococcus pneumoniae, buffered aqueous solution
N 5521
a2-(3,6)-Neuraminidase
From Clostridium perfringens (C. welchii), recombinant, expressed in E. coli,

N 8271
a2-(3,6,8,9)-Neuraminidase
From Arthrobacter ureafaciens, positionally specific, recombinant, expressed in E. coli,

N 3786
a2-(3,6,8,9)-Neuraminidase
Proteomics grade
N 4883
Neuraminidase Agarose
From Clostridium perfringens (C. welchii), type X-A, ammonium sulfate suspension,
20-30 units/g agarose
N 5254
Neuraminidase Agarose
From Clostridium perfringens (C. welchii), type VI-A, ammonium sulfate suspension
N 2133
Neuraminidase
From Clostridium perfringens (C. welchii), type X, lyophilized powder,

minimum 50 units/mg, 150-400 units/mg
N 2876
Neuraminidase
From Clostridium perfringens, 0.5-6 units/mg solid, From Vibrio cholerae, type III,
1-3 units/mg protein
N 5631
Neuraminidase
From Clostridium perfringens (C. welchii), type VIII, lyophilized powder, 10-20 units/mg
N 3001
Neuraminidase
From Clostridium perfringens (C. welchii), type VI, lyophilized powder,

2-5 units/mg protein (mucin), 6-10 units/mg
N 7771
Neuraminidase
From Salmonella typhimurium, recombinant, expressed in E. coli, lyophilized powder,

minimum 400 units/mg
N 3642
Neuraminidase
From Arthrobacter ureafaciens, buffered aqueous solution, minimum 8 units/ml
N 7885
Neuraminidase
From Vibrio cholerae, type III, 1-3 units/mg protein
N 6514
Neuraminidase
From Vibrio cholerae, type II, saline solution, 8-24 units/mg
C 6105
Novozyme 188
Liquid, minimum 250 units/g
P 7052
Pectin Lyase acidic enzyme
From Aspergillus niger, buffered aqueous glycerol solution, 50-150 units/mg
P 2611
Pectinase
From Aspergillus aculeatus, minimum 26,000 units/ml, Pectinex Ultra SPL
P 2736
Pectinase
From Aspergillus niger, minimum 3,000 units/ml, Pectinex 3X L
P 2401
Pectinase
from Rhizopus sp., crude powder, 400-800 units/g solid
P 0764
Pectinesterase
From orange peel, ammonium sulfate suspension, 50-350 units/mg
P 3026
Pectolyase
From Aspergillus japonicus, lyophilized powder, minimum 0.3 units/mg
P 5431
Pectolyase
From Aspergillus japonicus, lyophilized powder, minimum 2 units/mg
P 8375
Peroxidase, Horseradish
Essentially salt-free
G 5166
PNGase F
from Chryseobacterium (Flavobacterium) meningosepticum, buffered aqueous solution
P 3304
Polygalacturonase
From Aspergillus japonicus, ammonium sulfate suspension, 300-1,500 units/mg
P 5420
Pullulanase
From Klebsiella pneumoniae, ammonium sulfate suspension, minimum 5 units/mg
P 1067
Pullulanase
From Klebsiella pneumoniae, lyophilized powder, 10-30 units/mg
S 9754
Sulfatase
From abalone entrails, type VIII, lyophilized powder, 20-40 units/mg
S 1629
Sulfatase
From Aerobacter aerogenes, type VI, buffered aqueous glycerol solution,

10-20 units/ml, 2-5 units/mg
S 9626
Sulfatase
From Helix pomatia, type H-1, minimum 10,000 units/g solid
S 8504
Sulfatase
From Patella vulgata (keyhole limpet), type IV, essentially salt-free, lyophilized powder,
10 units/mg
Glycobiology
Product Directory
Neuraminidase
Pectinase
Pectolyase
Pullulanase
sigma-aldrich.com
Sulfatase
74
Enzymes
Sulfatase (cont)
S 8629
Sulfatase
From Patella vulgata (keyhole limpet), type V, Essentially salt-free, lyophilized powder,
minimum 5 units/mg
S 1062
2-O-Sulfatase
From Flavobacterium heparinum, buffered aqueous solution, 10-40 units/ml
S 9751
Sulfatase
From Helix pomatia, type H-2, crude solution, minimum 2,000 units/ml
T 4528
Thioglucosidase
From Sinapis alba (white mustard) seed, minimum 175 units/g
T 8778
Trehalase
From porcine kidney, buffered aqueous glycerol solution, minimum 0.4 units/mg
V 2010
Viscozyme L
From Aspergillus sp., enzyme mixture containing carbohydrases, including arabanase,

cellulase, B-glucanase, hemi-cellulase and xylanase
X 4001
Xylanase
From Aureobasidium pullulans, lyophilized powder, 50-200 units/mg
X 2753
Xylanase
From Thermomyces lanuginosus, powder, minimum 2,500 units/g
X 3876
Xylanase
From Trichoderma viride, lyophilized powder, 100-300 units/mg
X 1378
b1,4-Xylosidase
From Enterobacter aerogenes, recombinant, expressed in E. coli, buffered aqueous solution
X 3501
b-Xylosidase
Xylanase
Xylosidase
Glycobiology
Product Directory
GPI Anchor Enzymes

C 2557
Ceramide Glycanase
Buffered aqueous solution, minimum 0.01 units/mg
E 2277
Endoglycoceramidase II
plus activator II
From Rhodococcus sp., endo-type cleavage of cell surface glycosphingolipids of living cells
without damaging other cell components
P 5542
Phospholipase C,
Phosphatidylinositol-specific
From Bacillus cereus, lyophilized powder, 3,000 units/mg

Used for the release of GPI anchored proteins from the membrane
P 8804
Phospholipase C,
Phosphatidylinositol-specific
From Bacillus cereus, buffered aqueous glycerol solution, 3,000 units/mg

Used for the release of GPI anchored proteins from the membrane
P 5147
Pronase E
From Streptomyces griseus (protease), powder, type XIV, approx. 4 units/mg

A mixture of at least three proteolytic activities including an extracellular serine protease
sigma-aldrich.com
Glycosyltransferases
74188
a-1,3-Galactosyl-Transferase Kit
a-1,3-Galactosylation kit, contains glycosyltransferase, nucleotide sugar (donor), buffer

and supplementary reagents for performing five glycosylations
77038
a-1,3-Galactosyl-Transferase
Recombinant, expressed in E. coli, solution, ~0.5 units/ml

Enzyme employed for the chemoenzymatic synthesis of trisaccharides from disaccharides in
quantitative yield; synthesis of xenoactivea-galactosyl epitopes
59505
b-1,4-Galactosyl Transferase Kit
b-1,4-Galactosylation Kit
90261
b-1,4-Galactosyl Transferase I
Recombinant human, expressed in Saccharomyces cerevisiae

Enzyme catalyst for the stereo-selective synthesis of galactosyl carbohydrates
48279
From bovine milk, lyophilized powder, ~1 units/mg
48281
From bovine milk, lyophilized powder, ~8 units/g

Catalyst for the chemoenzymatic oligosaccharide synthesis, e.g. transfer of galactose
75
Inhibitors
Inhibitors of Carbohydrate Metabolism
P 7340
()-threo-1-Phenyl-2-decanoyl-amino-3-morpholino-1-propanol hydrochloride
Syn: PDMP
H 9632
(2R,5R)-Bis(hydroxymethyl)-(3R,4R)-dihydroxypyrrolidine
Syn: DMDP
D 8390
1,4-Dideoxy-1,4-imino-D-mannitol hydrochloride
Minimum 90% by HPLC
D 9160
1-Deoxymannojirimycin hydrochloride
Syn: 1,5-Dideoxy-1.5-imino-D-mannitol HCl
D 9305
1-Deoxynojirimycin hydrochloride
Syn: 1,5-Dideoxy-1,5-imino-D-sorbitol HCl
A 5416
2-Acetamido-1,2,5-trideoxy-1,5-imino-D-glucitol
Minimum 90% by HPAE
D 8875
2-Deoxy-D-glucose 6-phosphate sodium salt
Minimum 95% by TLC
Deoxyglucose
D 8375
2-Deoxy-D-glucose
Grade II, crystalline
D 6134
2-Deoxy-D-glucose
Grade III, crystalline
D 3179
2-Deoxy-D-glucose
SigmaUltra
B 1147
6-Bromo-4-cyclohexene-1,2,3-triol
Mixed isomers
B 6676
7(S)-Brefeldin A-7-3H
Ethanol solution
B 4894
Benzyl 2-acetamido-2-deoxy-a-D-galactopyranoside
Syn: a-D-GalNAc-1OCH2Ph
B 6542
Brefeldin A
From Penicillium brefeldianum,

for molecular biology, minimum 98% (TLC)
B 7651
Brefeldin A
From Penicillium brefeldianum,

minimum 99% (TLC)
C 3784
Castanospermine
From Castanospermum australe seeds
27689
Concanamycin A
From Streptomyces sp., BioChemika
C 9705
Concanamycin A
From Streptomyces sp., syn: Folimycin
C 5424
Conduritol B epoxide
Syn: 1,2-Anhydro-myo-inositol
D 9641
Deoxygalactonojirimycin hydrochloride
Syn: 1,5-Dideoxy-1,5-imino-D-galactitol
P 4194
DL-threo-1-Phenyl-2-palmitoyl-amino-3-morpholino-1-propanol
Syn: ()-PPMP, minimum 98% by TLC
69895
Monensin decyl ester, Selectophore
100 mg/ml in tetrahydrofurane
M 5273
Monensin sodium salt
Syn: Monensin A
D 9050
N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid
Approx. 95% by TLC
N 8028
Nikkomycin Z
From Streptomyces tendae
B 8299
N-Butyldeoxynojirimycin
Approx. 95% by TLC
M 1777
N-Methyl-1-deoxynojirimycin
Minimum 98% by HPLC
S 6640
Swainsonine
From Rhizoctonia leguminicola
S 9263
Swainsonine
Synthetic
T 0527
Tunicamycin A1 homolog
Approx. 95%
T 1152
Tunicamycin C2 homolog
Approx. 95% (HPLC)
T 7765
Tunicamycin
From Streptomyces sp.
Brefeldin
Glycobiology
Product Directory
Con A
Monensin
Nojirimycin
Swainsonine
sigma-aldrich.com
Tunicamycin
76
Substrates
Glycolytic Enzyme Substrates
Acetylneuraminic
M 8639
2-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid
Minimum 95% (HPLC), sodium salt hydrate
N 1266
2-O-(o-Nitrophenyl)-a-D-N-acetylneuraminic acid
Approx. 95%
N 1516
2-O-(p-Nitrophenyl)-a-D-N-acetylneuraminic acid
Approx. 95%
B 4666
5-Bromo-4-chloro-3-indolyl a-D-N-acetylneuraminic acid
Minimum 90%, sodium salt
Arabinofuranoside
N 3641
4-Nitrophenyl a-L-arabinofuranoside
N 3512
4-Nitrophenyl a-L-arabinopyranoside
Minimum 99% (TLC)
N 0520
4-Nitrophenyl b-L-arabinopyranoside
Minimum 98% (TLC)
Minimum 98% (TLC)
Cellobioside
N 4764
2-Nitrophenyl b-D-cellobioside
Minimum 95% (HPLC)
M 6018
4-Methylumbelliferyl b-D-cellobioside
Minimum 98% (TLC)
N 5759
4-Nitrophenyl b-D-cellobioside
Minimum 98% (HPLC)
B 6795
5-Bromo-4-chloro-3-indolyl b-D-cellobioside
Syn: X-Cellobiose
N 0145
4-Nitrophenyl b-D-cellopentaoside
Minimum 90%, powder
N 1262
4-Nitrophenyl b-D-cellotetraoside
Minimum 75%
N 4889
4-Nitrophenyl b-D-cellotrioside
Approx. 95%
M 9763
4-Methylumbelliferyl b-D-N,N-diacetylchitobioside
Minimum 98% (TLC), hydrate
N 6133
4-Nitrophenyl N,N-diacetyl-b-D-chitobioside
Minimum 99% (TLC)
N 8638
4-Nitrophenyl b-D-N,N,N-triacetylchitotriose
Minimum 98% (TLC)
M 5639
4-Methylumbelliferyl b-D-N,N,N-triacetylchitotrioside
Hydrate, catalyzed by chicken lysozyme, lipase
Chitobioside
Glycobiology
Product Directory
Fucopyranoside
N 3628
4-Nitrophenyl a-L-fucopyranoside
Minimum 98% (TLC)
N 3378
4-Nitrophenyl b-D-fucopyranoside
Minimum 98% (TLC)
N 2505
4-Nitrophenyl b-L-fucopyranoside
Minimum 99% (TLC)
B 9511
5-Bromo-4-chloro-3-indolyl b-D-fucopyranoside
Minimum 98%
B 2280
5-Bromo-4-chloro-3-indolyl b-L-fucopyranoside
Minimum 98%
N 3253
2-Nitrophenyl b-D-fucopyranoside
Minimum 99% (TLC)
M 5510
4-Methylumbelliferyl b-D-fucoside
Minimum 98%
M 4508
4-Methylumbelliferyl b-L-fucoside
Minimum 98%
Fucoside
sigma-aldrich.com
Galactopyranoside
67610
1-Methyl-3-indolyl-b-D-galactopyranoside
Minimum 98.0% (HPLC), BioChemika
N 7508
1-Naphthyl a-D-galactopyranoside
Minimum 99% (TLC)
N 2259
2-Naphthyl b-D-galactopyranoside
Minimum 99% (TLC)
N 2509
2-Nitrophenyl 1-thio-b-D-galactopyranoside
Minimum 99% (TLC)
N 8888
2-Nitrophenyl a-D-galactopyranoside
Minimum 99% (TLC)
N 1127
2-Nitrophenyl b-D-galactopyranoside
Minimum 98% (enzymatic)
N 6129
Minimum 98%
C 7553
4-Chloro-3-indolyl b-D-galactopyranoside
Minimum 99% (TLC)
M 7633
4-Methylumbelliferyl a-D-galactopyranoside
Minimum 98% (TLC)
M 1633
4-Methylumbelliferyl-b-D-galactopyranoside
Minimum 99% (TLC)
N 2257
4-Nitrophenyl 1-thio-b-D-galactopyranoside
Minimum 95%
N 2766
4-Nitrophenyl 2-acetamido-2-deoxy-3-O-(2-acetamido-2-deoxyb-D-glucopyranosyl)-a-D-galactopyranoside
Minimum 98%
N 3016
4-Nitrophenyl 2-acetamido-2-deoxy-3-O-(b-D-galactopyranosyl)a-D-galactopyranoside
Minimum 98%
N 7643
4-Nitrophenyl 6-O-b-D-galactopyranosyl-b-D-galactopyranoside
Minimum 95%
N 0877
Minimum 99% (TLC)
77
Substrates
Galactopyranoside (cont)
N 1252
4-Nitrophenyl b-D-galactopyranoside
Minimum 98%
T 3661
4-Trifluoromethylumbelliferyl b-D-galactopyranoside
Minimum 98% (TLC)
B 2904
5-Bromo-3-indolyl b-D-galactopyranoside
Molecular biology grade
B 4387
5-Bromo-3-indolyl b-D-galactopyranoside
Minimum 98%
B 6024
5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside
5 mg tablet
B 9146
Minimum 98%
B 4252
Minimum 98%, powder
16669
5-Bromo-6-chloro-3-indolyl-b-D-galactopyranoside
98.0% (HPLC), BioChemika
57897
5-Iodo-3-indolyl-b-D-galactopyranoside
36931
6,8-Difluoro-4-methylumbelliferyl-b-D-galactopyranoside
98.0% (TLC), BioChemika
24904
6-Chloro-3-indolyl-b-D-galactopyranoside
F 2756
Fluorescein di-(b-D-galactopyranoside)
Minimum 98%
F 6542
Fluorescein mono-b-D-galactopyranoside
Approx. 90%
I 7513
Indoxyl b-D-galactopyranoside
Minimum 98%
N 2885
Naphthol AS-BI b-D-galactopyranoside
Minimum 99% (TLC)
N 0257
2-Nitrophenyl N-acetyl-a-D-galactosaminide
Minimum 99% (TLC)
N 3273
2-Nitrophenyl-N-acetyl-b-D-galactosaminide
Minimum 99% (TLC)
M 9659
4-Methylumbelliferyl N-acetyl-b-D-galactosaminide
Minimum 98% (TLC)
N 4264
4-Nitrophenyl N-acetyl-a-D-galactosaminide
Minimum 98%
N 9003
4-Nitrophenyl N-acetyl-b-D-galactosaminide
Minimum 98%
B 3166
5-Bromo-4-chloro-3-indolyl N-acetyl-b-D-galactosaminide
Approx. 95%
Glycobiology
Product Directory
Galactosaminide
Galacturonide
N 8755
4-Nitrophenyl b-D-galacturonide
Minimum 98% (TLC)
Glucopyranoside
N 2007
2-Naphthyl b-D-glucopyranoside
N 8016
2-Nitrophenyl b-D-glucopyranoside
Minimum 99% (TLC)
M 9766
4-Methylumbelliferyl a-D-glucopyranoside
Minimum 99% (TLC)
Minimum 98% (TLC)
M 3633
4-Methylumbelliferyl b-D-glucopyranoside
Minimum 99% (TLC)
M 0662
4-Methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxyb-D-glucopyranoside)
Approx. 98% (TLC), sodium salt
N 2381
4-Nitrophenyl 1-thio-b-D-glucopyranoside
Approx. 95%
N 5513
4-Nitrophenyl 2-acetamido-2-deoxy-3-O-b-D-galactopyranosylb-D-glucopyranoside
Minimum 98% (TLC)
N 1377
4-Nitrophenyl a-D-glucopyranoside
Minimum 99%
N 7006
4-Nitrophenyl b-D-glucopyranoside
Minimum 98% (TLC)
B 4527
5-Bromo-4-chloro-3-indolyl b-D-glucopyranoside
Approx. 98%
36937
6,8-Difluoro-4-methylumbelliferyl-b-D-glucopyranoside
98.0% (TLC), BioChemika
F 4521
Fluorescein di-(b-D-glucopyranoside)
Minimum 90%
M 9881
4-Methylumbelliferyl N-acetyl-a-D-glucosaminide
Minimum 98% (TLC)
M 2133
4-Methylumbelliferyl N-acetyl-b-D-glucosaminide
Minimum 98% (TLC), dihydrate
N 8759
4-Nitrophenyl N-acetyl-a-D-glucosaminide
Minimum 98%
N 9376
4-Nitrophenyl N-acetyl-b-D-glucosaminide
98-100%
B 3041
5-Bromo-4-chloro-3-indolyl N-acetyl-b-D-glucosaminide
Minimum 98%
N 4006
Naphthol AS-BI N-acetyl-b-D-glucosaminide
Minimum 95% (TLC)
I 6893
Indoxyl b-D-glucoside
SigmaUltra, minimum 97%
I 3750
Indoxyl b-D-glucoside
Minimum 97%
Glucoside
sigma-aldrich.com
Glucosaminide
78
Substrates
Glucuronide
N 9013
1-Naphthyl b-D-glucuronide
N 2645
2-Nitrophenyl b-D-glucuronide
Minimum 98% (TLC), potassium salt
M 9130
4-Methylumbelliferyl b-D-glucuronide
Minimum 98% (TLC), hydrate
M 5664
4-Methylumbelliferyl b-D-glucuronide
SigmaUltra, hydrate
N 1627
4-Nitrophenyl b-D-glucuronide
Minimum 98% (TLC)
T 6410
4-Trifluoromethylumbelliferyl glucuronide
Minimum 98% (TLC), potassium salt
B 8049
5-Bromo-4-chloro-3-indolyl b-D-glucuronide
10 mg tablet, cyclohexylammonium salt
B 0522
Minimum 98%, cyclohexylammonium salt
B 3783
B 6650
Minimum 98% (TLC), cyclohexylammonium salt
B 8174
10 mg tablet, sodium salt
B 5285
Minimum 98%, sodium salt
B 4782
Minimum 98% (TLC), sodium salt
B 4532
B 4657
36939
6,8-Difluoro-4-methylumbelliferyl-b-D-glucuronide
BioChemika, for fluorescence, 98.0% (TLC), lithium salt
24907
6-Chloro-3-indolyl-b-D-glucuronide
BioChemika, 97.0% (HPLC), cyclohexylamine salt
I 7638
Indoxyl b-D-glucuronide
Cyclohexylammonium salt
N 1875
Naphthol AS-BI b-D-glucuronide
Approx. 95%, sodium salt
P 0376
Phenolphthalein b-D-glucuronide
Sodium salt
P 0501
Phenolphthalein b-D-glucuronide
Free acid
Approx. 99%, sodium salt
Glycobiology
Product Directory
Lactopyranoside
M 2405
4-Methylumbelliferyl b-D-lactopyranoside
Minimum 95% (TLC)
N 1752
4-Nitrophenyl b-D-lactopyranoside
Minimum 99% (TLC)
Maltopentaoside
N 1519
4-Nitrophenyl-a-D-maltopentaoside
Minimum 98%
N 5885
4-Nitrophenyl a-D-maltoside
Minimum 99% (TLC)
N 1884
4-Nitrophenyl b-D-maltoside
Minimum 99% (TLC)
Maltoside
Mannopyranoside
M 3657
4-Methylumbelliferyl a-D-mannopyranoside
Approx. 98% (TLC)
M 0905
4-Methylumbelliferyl b-D-mannopyranoside
Minimum 98% (TLC)
N 2127
4-Nitrophenyl a-D-mannopyranoside
Minimum 98% (TLC)
N 1268
4-Nitrophenyl b-D-mannopyranoside
Approx. 98%
B 4526
5-Bromo-4-chloro-3-indolyl a-D-mannopyranoside
Approx. 95%
G 2000
2-(b-D-Galactosidoxy)naphthol AS-LC
Minimum 95% (TLC)
A 8750
N-Acetyl-b-D-glucosamine naphthol AS-LC
Minimum 95% (TLC)
D-()-Salicin
Minimum 99%, substrate for b-Galactosidase
Naphthol
Salicin
S 0625
Rhamnopyranoside
sigma-aldrich.com
N 7763
4-Nitrophenyl a-L-rhamnopyranoside
Approx. 99% (TLC)
Xylopyranoside
N 3629
2-Nitrophenyl b-D-xylopyranoside
Minimum 99% (TLC)
M 7008
4-Methylumbelliferyl-b-D-xylopyranoside
Minimum 98% (TLC)
N 1895
4-Nitrophenyl a-D-xylopyranoside
Minimum 98% (TLC)
N 2132
4-Nitrophenyl b-D-xylopyranoside
Minimum 98% (TLC)
79
Neoglycoproteins
Neoglycoproteins
Cellulose
A 8585
Cellobiose-BSA
Galactose
A 5908
2-Amido-GalN-BSA
A 2420
a-Gal-FITC-BSA
A 6544
a-Gal-TRITC-BSA
A 8165
b-Gal-FITC-BSA
A 1159
Aminophenyl-b-GalNAc-BSA
Glucose
A 6158
GlcNAc-BSA
A 5543
a-Glc-FITC-BSA
A 1034
Aminophenyl-b-GlcNAc-BSA
A 7172
b-Glc-FITC-BSA
A 6052
GlcNAc-resorufin-BSA
Fructose
A 8426
Fructosamine-BSA (glycated)
A 8301
Fructosamine-HSA (glycated)
A 4042
1-Amido-Fuc-biotin-BSA
A 6033
1-Amido-Fuc-BSA
A 5793
a-L-Fuc-FITC-BSA
A 5918
a-L-Fuc-TRITC-BSA
Glycobiology
Product Directory
Fucose
Lactose
A 8210
Lactose-BSA
A 7799
b-Lactose-biotin-BSA
A 8040
b-Lactose-FITC-BSA
A 7665
b-Lactose-TRITC-BSA
A 5783
Lactitol-1-N-BSA
Maltose
A 5283
Maltitol-1-N-BSA
A 8460
Maltose-BSA
Mannose
A 7924
a-Man-biotin-BSA
A 8303
a-Man-BSA
A 7790
a-Man-FITC-BSA
A 7915
a-Man-TRITC-BSA
A 4664
Aminophenyl-a-Man-BSA
A 3955
b-Xyl-FITC-BSA
sigma-aldrich.com
Xylose
80
Lectins
Lectins are proteins or glycoproteins from non-immune origins that agglutinate cells and/or precipitate complex carbohydrates. The agglutination activity of these highly specific carbohydrate-binding molecules is usually inhibited by a
simple monosaccharide, but for some lectins di-, tri-, and even polysaccharides are required.
Sigma offers a wide range of lectins suitable for the following applications:
Carbohydrate studies
Lymphocyte subpopulation studies
Blood group typing
Fractionation of cells and other particles
Mitogenic stimulation
Histochemical studies
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Glycobiology
Product Directory
Lectins are isolated from a wide variety of natural sources. Sigma offers a range of lectins suitable for all of the
applications noted above.
Common Name
Taxonomic Name
Common Name
Taxonomic Name
Abrin
Abrus precatorius
Labumum, Scotch
Labumum polyphemus
Asparagus pea
Tetragonolobus purpureas
Lentil
Lens culinaris
Avocado
Perseau americana
Lotus
Bitter pear melon
Momordica charantia
Mistletoe, European
Viscum album
Broad bean
Vicia faba
Mung bean
Vigna radiata
Camels foot tree
Bauhinia purpurea
Mushroom
Agaricus bisporus
Castor bean
Ricinus communis
Osage orange
Maclura pomifera
Chick pea
Cicer arietinum
Pagoda tree
Sophora japonica
Cobra, Mozambique
Naja mocambiqu mocambique
Pea, garden
Pisum sativum
Cobra, Thailan
Naja naja kaouthis
Peanut
Arachis hypogaea
Con A
Canavalia ensiformis
Pokeweed
Phytolacca americana
Concanavalin A
Potato
Solanum tuberosum
Coral tree, Israel
Erythrina corallodendron
Red kidney bean
Phaseolus vulgaris
Daffodil
Narcissus pseudonarcissus
Red marine algae
Ptilota plumosa
Eel
Anguilla anguilla
Roman snail
Helix pomatia
Elder
Sambucus nigra
Scarlet runner bean
Phaseolus coccineus
Fava bean
Vicia faba
Siberian pea tree
Caragana arborescens
Furze
Ulex europaeus
Snail, garden
Helix aspersa
Gorse
Ulex europaeus
Snowdrop
Galanthus nivalis
Green marine algae
Codium fragile
Soybean
Glycine max
Hairy vetch
Vicia villosa
Spindle tree
Euonymus europaeus
Horse gram
Dolichos biflorus
Sweet pea
Lathyrus odoratus
Horseshoe crab
Limulus polyphemus
Thorn Apple
Datura stramonium
Jacalin
Artocarpus intefrifolia
Tomato
Lycopersicon esculentum
Jack bean
Wheat germ
Triticum vulgaris
Japanese wisteris
Wisteria floribanda
Winged bean
Psophocarpus tetragonolobus
Jequity bean
Abrus precatorius
Winged pea
Jimson weed
Datura stramonium
Many lectins are available as conjugates. Conjugation does not alter the specificity of the lectin. The degree of conjugation
is expressed as 1) a mole of dye per mole of lectin basis 2) a unit of enzyme activity per mg of protein or 3) a mg of
protein per ml of packed gel basis. For colloidal gold conjugates, the activity of the lectin is determined by a dot blot
assay using an appropriate glycoprotein. Agglutination activity is expressed in microgram per ml and is determined
from serial dilution studies of a 1 mg per ml solution. This activity is the lowest concentration of lectin to agglutinate
a 2% suspension of appropriate erythrocytes after 1 hour incubation at 25 C.
81
Lectins
Lectins
Product
Number
Source
Mol. Wt. (kDa)
Subunits
Specificity
Blood Group
Specificity Sugar
L 5640
Agaricus bisporus
58.5
b-Gal(13)GalNAc
L 0881
Arachis hypogaea
120
b-Gal(13)GalNAc
42
a-GalOMe
114
A,B
a-Gal, a-GalNAc
114
a-Gal
L 7759
Peroxidase
L 6135
Biotin
L 7381
FITC
L 3766
L 3515
Mitogenic
Activity
TRITC
Artocarpus integrifolia
L 4650
Peroxidase
L 5147
Agarose
(+)
BS-I
L 3759
Biotin
L 9381
FITC
L 5264
TRITC
L 0890
FITC
L 3019
BS-I-B4
L 5391
Peroxidase
L 2140
Biotin
L 2895
FITC
L 9637
Caragana arborescens
60;120(c)
2;4
GalNAc
L 3141
Cicer arietinum
44
fetuin
L 2638
Codium fragile
60
GalNAc
C 7275
Concanavalin A
102
a-Man, a-Glc
(+)
L 6397
Peroxidase
C 2272
Biotin
C 7642
FITC
C 6904
Agarose
C 9017
Sepharose
L 5021
Gold, 10 nm
L 3642
Gold, 20 nm
C 7898
Ferritin
51
a-Man, a-Glc
(+) (d)
L 9385
Succinyl-Concanavalin A
FITC
L 2766
Datura stramonium
86
2(a and b)(a)
(GlcNAc)2
L 2785
Dolichos biflorus
140
A1
a-GalNAc
L 1287
Peroxidase
L 6533
Biotin
L 9142
FITC
L 9658
TRITC
L 9894
Agarose
L 5390
Erythrina cristagalli
56.8
2(a and b)(a)
b-Gal(14)GlcNAc
L 7400
Euonymus europaeus
166
4(a and b)(a)
B,H
a-Gal(13)Gal
L 8725
Galanthus nivalis
52
(h)
non-reduc. D-Man
L 8775
Agarose
(+)
sigma-aldrich.com
L 3885
Glycobiology
Product Directory
Bandeiraea simplicifolia
L 2380
82
Lectins
Lectins
Product
Number
Source
Mol. Wt. (kDa)
Subunits
Specificity
Blood Group
Specificity Sugar
Mitogenic
Activity
L 1395
Glycine max
110
GalNAc
(+) (b)
L 2650
Peroxidase
L 4511
TRITC
79
GalNAc
79
GalNAc
49
a-Man
18
NeuNAc
L 6635
L 8764
Biotin
L 3382
Helix pomatia
L 6387
Peroxidase
L 6512
Biotin
L 1034
FITC
L 1261
L 9267
TRITC
Lens culinaris
L 4143
Biotin
L 9262
FITC
L 0511
Limulus polyphemus
400
L 2886
Lycopersicon esculentum
71
L 0401
L 8025
L 4401
L 3138
L 4389
(+)
Agarose
L 2263
L 0651
Glycobiology
Product Directory
Helix aspersa
(GlcNAc)3
(+) (e)
sialic acid
(+)
Biotin
FITC
Maackia amurensis
130
2(a and b)
112
128
oligosaccharide
(+)
126
oligosaccharide
(+)
(GlcNAc)3
(+)
a-Man
(+)
Peroxidase
Phaseolus coccineus
TRITC
Phaseolus vulgaris
L 8629
L 6139
PHA-E
TRITC
L 2769
PHA-L
L 8754
PHA-P
L 2646
PHA-M
L 9379
Phytolacca americana
32(f)
L 5380
Pisum sativum
49
L 0770
4(a and b)(a)
35
GalNAc, Gal
120
b-Gal
FITC
L 9895
Pseudomonas aeruginosa (PA-I) 13-13.7
L 2138
Psophocarpus tetragonolobus
L 3139
Peroxidase
L 3014
Biotin
L 3264
Gal
FITC
Ricinus communis
sigma-aldrich.com
L 7886
Agglutinin, RCA120
L 2758
Peroxidase
L 9514
Ricin, A chain
L 4022
Ricin, A chain, deglycosylated
L 9639
Ricin, B chain
L 6890
Sambucus nigra
L 4266
Solanum tuberosum
83
Lectins
Lectins
Product
Number
Source
Mol. Wt. (kDa)
Subunits
Specificity
Blood Group
Specificity Sugar
L 9254
120(A), 58(B),
117(C)
4;2;4
a-L-fuc
36
L 5759
Peroxidase
L 3134
Biotin
L 5644
FITC
L 9640
Triticum vulgaris
L 3892
Peroxidase
L 0390
Peroxidase (inactive)
L 5142
Biotin
L 4895
FITC
L 9884
Evans Blue
L 1894
Gold, 10 nm
L 1882
Agarose
(GlcNAc)2, NeuNAc
Mitogenic
Activity
(+)
L 8146
Peroxidase
L 8262
Biotin
L 9006
FITC
L 4889
TRITC
L 4011
Vicia villosa
L 9388
Agarose
L 7513
Isolectin B4
L 7888
Agarose
L 2662
68
a-L-Fuc
139
4(a)
A1+Tn
GalNAc
143
Tn
GalNAc
Viscum album
115 g
4(a and b)(a)
b-Gal
Wisteria floribunda
68
GalNAc
34
GalNAc
L 1516
Biotin
L 2016
Reduced
L 1766
Biotin
Notes:
a Subunits are of different molecular weights
b Mitogenic for neuraminidase-treated lymphocytes
c Concentration-dependent mol. wt. change
d Non-agglutinating and mitogenic
e Inhibits mitogenic activity of PHA

f Data given for PWM Pa2
h Agglutinates rabbit, but not human, erythrocytes
sigma-aldrich.com
UEA I
Glycobiology
Product Directory
Ulex europaeus
L 5505
88
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Glycosylation Methods and Analysis Sigma

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Glycosylation Methods and Analysis Sigma

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Glycoprotein

during MS analysis due to the heterogeneity of the

Chemical Deglycosylation Strategies

Glycan Labeling and Analysis

Glycan and Carbohydrate Standards

Glycobiology Product Directory

or threonine. GPI-anchored proteins are

Key to Monosaccharide Symbols, Abbreviations, and Projections

3-D Chair projection

Key to Monosaccharide Symbols, Abbreviations, and Projections

3-D Chair projection

Key to Monosaccharide Symbols, Abbreviations, and Projections

3-D Chair projection

a-L-Iduronic acid (IdoA)

b-D-Glucuronic acid (GlcA)

Classification and Structure of Glycan Components

Inhibition or elimination of glycosylation in the study of

Basic N-linked Structure

Complex Structure (tetraantennary)

related to cell signaling events in the cell. O-Linked glycans

Di- and Trisialated O-Linked Core

O-Linked Core 2 Hexasaccharide

GPI Anchor Glycoproteins

Their functionality ranges from enzymatic to antigenic

Purification of proteins selectively utilizing their glycan

m-Aminophenylboronic Acid Matrices

Matrix: cross-linked 6% beaded agarose

Matrix: acrylic beads

Matrix: 6% beaded agarose

(For additional Lectins see page 80)

Matrix: 4% beaded agarose

3. Romero, P.A., et al., Glycoprotein biosynthesis in Saccharomyces

Wheat Germ (Triticum vulgaris) matrices are specific for

Wheat Germ (Triticum vulgaris)

Matrix: Cross-Linked 4% beaded agarose

Wheat Germ (Triticum vulgaris)

Matrix: 6% agarose macrobeads

Initial detection of glycoproteins in vitro is routinely accomplished on SDS-PAGE gels and

Glycoprotein Detection Kit

from horseradish, reported as having a carbohydrate

Time for gel thickness 0.5-0.75 mm or for membrane

Time for gel thickness 1.0-1.5 mm

1-2 hours or until bands turn magenta

1-2 hours or until bands turn magenta

Band color will intensify with changes of fresh water

Band color will intensify with changes of

Colorimetric Detection on PAGE and

Ovalbumin (glycosylated and phosphorylated)

The classical method for in-gel carbohydrate detection

BSA (not glycosylated, not phosphorylated)

Sufficient reagents are supplied for 10 mini gels.

IgG Heavy Chain (glycosylated)

IgG Light Chain

ProteoProfile PTM Marker

Figure 2. Mouse IgG and rabbit IgG were separated on a 420%

Optimal results are obtained on PDF membranes.

Biotin Labeled Glycan

Detection of Biotin Labeled

CH2 = CH2 = CH2 = CH2 = C = N = NH2

Streptavidin, Peroxidase Labeled

Sodium (meta) Periodate

Tetramethylbenzidine Liquid Substrate System

Chemiluminescent Peroxidase Substrate 60 ml

Chemiluminescent Peroxidase Substrate 120 ml