Chromotek-GFP-Trap®, a GFP-binding protein
based on a single domain antibody derived from Lama alpaca.
A genius for quantitative immunoprecipitation
of GFP fusion proteins.
G reen fluorescent proteins (GFP)
and variants thereof are widely
used to study protein localization and
dynamics. However, among the most
commonly used tags for immuno-
precipitation (a brief review in Box
1), the use of GFP is limited due to
previously available anti-GFP antibod-
ies, either polyclonal or monoclonal,
not being comparable to those against
other tags.
GFP-Trap® is a high quality GFP-
binding protein based on a single
domain antibody derived form Lama
Alpaca. It is characterized by a small
barrel shaped structure (13 KDa, 2.5
nm X 4.5 nm) and a very high stability
(stable up to 70°C, functional within 2
M NaCl or 0.5% SDS). From detailed
in vitro binding analysis, we deter-
mined that one molecule GFP-Trap®
binds one molecule GFP in a stable
stoichiometric complex with a disso-
ciation constant (Kd) of 0.59 nM.
After coupling to monovalent matri-
ces (e.g. agarose beads or magnetic
particles), GFP-Trap® can be a robust
Box 2 | Comparative Immunoprecipitation Assay
1) GFP-Trap allows a very fast (~ 5 – 30 min) depletion of GFP from tested samples, which cannot
be achieved with conventional antibodies even after 12 h of incubation.
2) Only the antigen (GFP) was detectable on a coomassie gel , there is no heavy chain and light
chain
Allele Biotech-Introducing Cost Effectiveness to Research
Box 1 | Tags for Immunoprecipitation
To achieve effective immunoprecipitation, a researcher must first overcome the
difficulty of finding usable antibodies against a target of interest. Using tags that are
fused to the C- or N-terminus of the target protein is common practice. In general,
while being mindful of the unique nuances with each biological system, choosing
tags that have been tested in many situations and been proven to be non-interfer-
ing is ideal. The most commonly used tags are as follows: FLAG, Myc, HA, V5, T7,
and His; these are quite small in size and in theory less likely to interfere. GST and
GFP are well documented to form self-contained and stable structures independent
of their fusion partners and proven to not interfere in many cases despite their larger
size (in between 20-30kD). A top choice for pulldown experiments, GST can bind to
glutathione beads directly. GFP/RFP and their variants are excellent tags, having
the advantage of being a visualization module to follow the protein both inside cells
and during pulldown. With much greater stability, specificity, and affinity than previ-
ously available polyclonal or monoclonal anti-GFP/RFP antibodies, GFP-Trap® and
RFP-Trap®, the recent addition to antibodies for immunoprecipitation should make
GFP/RFP the most suitable tag for immunoprecipitation assays.
tool for:
* Identify interacting proteins * RIP assays (RNA immunoprecipita-
tion)
* Measure enzyme activity
* CLIP assays (in vivo Cross-Linking
* Determine DNA or RNA binding and ImmunoPrecipitation)
* Map DNA or RNA binding sites
With much greater stability, specificity,
* ChIP-CHIP assays and affinity, GFP-Trap®, the recent
addition to antibodies for immunopre-
cipitation, should make GFP the most
suitable tags for immunoprecipitation
assays. Direct comparison of the GFP-
Trap® with conventional antibodies is
shown in Box 2.
Besides the original avGFP from jelly
fish, GFP-Trap® can also bind to the
GFPS65T and eGFP versions and as
well as YFP and eYFP. It recognizes
and binds a three dimensional epitope
at the beta barrel structure. It does
not bind to CFP due to an amino acid
change within the recognized epit-
ope. GFP-Trap® does not recognize
unfolded or denatured GFP (e.g. on
Western blots).
DsRed, which is from corral instead
of jelly fish, is not recognized by GFP-
Trap. For binding DsRed-derived FPs
such as the commonly used mCherry,
mOrange, mPlum, mRuby and mRFP,
RFP-Trap® should be used.
Product List and Content
GFP-Trap® Beads and RFP-Trap® Beads:
A VHH domain binding protein derived from camelid heavy chain-only antibodies is coupled to agarose or magnetic beads for
immunoprecipitation. Typically, 1ul resin can effciently bind 1-3 µg proteins. Four unit sizes (0.5 ml, 2.5 ml, 5.0 ml and 10 ml)
are available. 0.5 ml size is enough for 20 RXNs.
GFP-Trap® Kits and RFP-Trap® Kits:
In addition to the VHH domain binding protein derived from camelid heavy chain-only antibodies being coupled to agarose or
magnetic beads for immunoprecipitation, precipitation buffers and a monoclonal anti-GFP antibody are provided for user con-
venience (0.5ml beads + 10ml lysis buffer + 50ml dilution buffer + 20ml wash buffer (NaCl 500mM) + 100µg monoclonal
anti-GFP antibody), which is enough for 20 RXNs.
GFP-Trap® Proteins and RFP-Trap® Proteins:
Uncoupled VHH domain binding protein derived from camelid heavy chain-only antibodies is provided for custom-conjugation
or other assays. The concentration is 1 µg/µl.

GFP-Trap® Beads RFP-Trap® Beads

GFP-Trap® Beads (Agarose) ACT-CM-GFA0050 0.5ml RFP-Trap® Beads (Agarose) ACT-CM-RFA0050 0.5ml
GFP-Trap® Beads (Agarose) ACT-CM-GFA0250 2.5ml RFP-Trap® Beads (Agarose) ACT-CM-RFA0250 2.5ml
GFP-Trap® Beads (Agarose) ACT-CM-GFA0500 5.0ml RFP-Trap® Beads (Agarose) ACT-CM-RFA0500 5.0ml
GFP-Trap® Beads (Agarose) ACT-CM-GFA1000 10.0ml RFP-Trap® Beads (Agarose) ACT-CM-RFA1000 10.0ml
GFP-Trap® Beads (Magnetic) ACT-CM-GFM0050 0.5ml RFP-Trap® Beads (Magnetic) ACT-CM-RFM0050 0.5ml
GFP-Trap® Beads (Magnetic) ACT-CM-GFM0250 2.5ml RFP-Trap® Beads (Magnetic) ACT-CM-RFM0250 2.5ml
GFP-Trap® Beads (Magnetic) ACT-CM-GFM0500 5.0ml RFP-Trap® Beads (Magnetic) ACT-CM-RFM0500 5.0ml
GFP-Trap® Beads (Magnetic) ACT-CM-GFM1000 10.0ml RFP-Trap® Beads (Magnetic) ACT-CM-RFM1000 10.0ml

GFP-Trap® Kits RFP-Trap® Kits

GFP-Trap® Kit (Agarose) ACT-CM-GFAK020 20 RXNs RFP-Trap® Co-IP Kit (Agarose) ACT-CM-RFAK020 20 RXNs
GFP-Trap® Kit (Magnetic) ACT-CM-GFMK020 20 RXNs RFP-Trap® Co-IP Kit (Magnetic) ACT-CM-RFMK020 20 RXNs

GFP-Trap® Protein ACT-CM-GFPTRAP 250µl, 1µg/µl RFP-Trap® Protein ACT-CM-RFPTRAP 250µl, 1µg/µl

GFP-Trap® Booster:
A VHH domain binding protein derived from camelid heavy chain-only antibodies is coupled to a strong fluorescent dye to both
stabilize GFP and enhance its fluorescence signals.

GFP-Trap® Booster ABP-CM-GBOOSTR 100ug

FP-TrapTM Binding Control

Blocked agarose beads or blocked magnetic particles, as binding controls.

FP-TrapTM Binding Control

Blocked agarose beads ACT-CM-BDCTRLA 0.5ml
Blocked magnetic particles ACT-CM-BDCTRLM 0.5ml

Conventional Anti-GFP/RFP antibodies:

Monoclonal or polyclonal antibodies against GFP/RFP are provided for post-precipitation detection or as controls.
Conventional Anti-GFP Antibodies
Mouse monoclonal antibody to GFP ABG-MP-MMGFP10 100ug, IgG1, for WB, ELISA, IP
Rabbit polyclonal antibody to GFP ABP-PAB-PAGFP10 100ug, IgG, for WB, ELISA, IP
Rat monoclonal [3H9] to GFP ACT-CM-MRGFP10 100ug, IgG2a , for WB, ELISA, IP
Rat monoclonal [5F8] to RFP ACT-CM-MRRFP10 100ug, IgG2a, for WB, ELISA, IP

Allele Biotech-Introducing Cost Effectiveness to Research

Protocols
Storage: at 2-8°C, do not freeze.
Cell Lysis
1. For one immunoprecipitation reaction resuspend cell pel-
let (~10^7 cells) in 200µl lysis buffer by pipetting (or using
a syringe)
2. Place the tube on ice for 30 min with extensively pipetting
every 10 min.
3. Spin cell lysate at 20.000x g for 5 -10 minutes at 4°C
4. Transfer supernatant to a pre-cooled tube. Adjust volume
with dilution buffer to 500µl–1000µl. Discard pellet
The cell lysate can be frozen at this point for long-term stor-
age at minus 80°C.
For immunoblot analysis dilute 50µl cell lysate with 50µl 2x
SDS-sample buffer. (-> refer as input)
Technical Notes:
Different buffers can be used to disrupt cells, for exmaple
higher salt or DNase I containing buffer to release chro-
matin proteins; RIPA buffer to release membrane bound
proteins ; different concentrations and combinations of
zwitterionic detergents to further optimize the sample
preparation.
Compared to commonly used ionic detergent such as SDS,
or non-ionic detergent such as Triton X-100, a less know
group called zwitterionic detergents contain both a posi-
tive and negative charge in their hydrophilic head group.
These compounds are electrically neutral like the nonionic
detergents, but can efficiently disrupt most protein-protein
interactions like the ionic detergents. For co-IP, non-specific
protein aggregation is to be disrupted, whereas specific,
stronger protein-protein interactions are to be preserved.
By using different concentrations and combinations of zwit-
terionic detergents, or sugar-derived polar head-containing
detergents, efficient cell lysis and protein extraction may be
achieved for co-IP. However, there is unfortunately no one
solution that works well for all cell types, proteins, or co-
IP requirements. A common buffer as recommended in the
GFP-Trap protocol, or an available system like AlleleExtract-
M should provide you with a good starting point.
GFP-Trap® Binding
5. Equilibrate GFP-Trap® beads in dilution buffer. Resus-
pend 20 - 30µl Beads Slurry in 500µl ice cold dilution buffer
and spin down at 2700x g for 2 minutes at 4°C. Discard
supernatant and wash binder two more times with 500µl ice
cold dilution buffer.
6. Add cell lysate to equilibrated GFP-Trap®_A beads
7. Incubate with gentle end-over-end mixing for 10 min – 2 h
at room temperature or 4°C
8. Spin tube at 2000x g for 2 minutes at 4°C
9. For western blot analysis dilute 50 µl supernatant with 50
µl 2x SDS-sample buffer (-> refer as non-bound)
10. Discard remaining supernatant
11. Wash pellet two times with 500 µl ice cold dilution buffer
(Optional: increase salt concentration in the second washing
step up to 500 mM)
Technical Notes:
Binding Capacity: typically 1ul resin can effciently bind
1-3ug proteins. The actual binding amount will depend on
different proteins.
Elution
12. Resuspend GFP-Trap®_A beads in 100 µl 2x SDS-
Sample buffer
13. Boil resuspended beads for 10 minutes at 95°C to dis-
sociate the immunocomplexes from the beads. The beads
can be collected by centrifugation at 2700x g for 2 minutes
at 4°C and SDS-PAGE is performed with the supernatant.
(-> refer as bound)
14. Optional: elute bound proteins by adding 50 µl 0.1 M
glycine pH 2.5 (incubation time: 30 sec – 2 min) followed by
neutralisation with 5 µl 1M Tris-base.
Technical Notes:
To elute native complex, the best way is acidic elution us-
ing 0.1 M glycine pH2.5 combined with a very fast addition
of 1 M Tris-base for neutralization.

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Suggested Buffers (as tested in our laboratory)
Lysis-buffer (for CoIP):
10 mM Tris/Cl pH7.5
150 mM NaCl
0.5 mM EDTA
0.5% NP40
1 mM PMSF freshly added (optional)
1x mammalian Protease Inhibitor Cocktail (e.g. Serva®)
freshly added
(optional for nuclear proteins / chromatin proteins:
DNaseI final conc. 1 μg/μl
2.5 mM MgCl2)
Dilution-buffer
10 mM Tris/Cl pH7.5
150 mM NaCl
0.5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva) freshly added
Camelid Antibody
C amelidae sp. family animals such as Camel and Llama
generate antibodies composed of heavy chains only.
The variable domain of camelid antibody heavy chain is
the smallest single domain antigen-binding fragment. The
~15kD fragment, term “nanobody” can recognize specific
antigens extremely well and bind very tightly. Combined with
its small size and ease for production in E. coli, the single
domain Camelid antibody fragment presents unprecedented
possibilities in live cell imaging as well as target molecule
isolation.
Generating camelid antibodies starts with creating a dis-
play library of variable domain VHH from immunized Llama,
then screening displaying phages or cells to find clones that
express nanobodies against the specific antigen with high
affinity and specificity, and cloning the gene encoding the
antigen-binding fragment to be expressed in E. coli.
Smaller antibody fragments have been tested for therapeu-
tic uses since classical IgG antibodies are too large to pene-
trate tissues well and are costly to produce. Different combi-
nations of antigen-binding variable regions have been used
(e.g., scFv, Fab, diabody) with varying degree of success.
By comparison, the N-terminal domain of camelid antibod-
ies, termed VHH domain (nanobody), represents a naturally
evolved, fully functional target binding fragment with many
advantages-one being that it is only 13-15 kD in size.
Allele Biotech-Introducing Cost Effectiveness to Research
Wash-buffer
10 mM Tris/Cl pH7.5
150 - 500 mM NaCl
0.5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) freshly added
RIPA-Buffer (for cell lysis):
10 mM Tris/Cl pH7.5
150 mM NaCl
0.1% SDS
1% TX100
1% Deoxycholate
5 mM EDTA
1 mM PMSF freshly added (optional)
1x Protease Inhibitor Cocktail (e.g. Serva®) freshly added
The only other known species outside the camelid family that
has heavy chain alone antibodies is the cartilaginous nurse
shark. Although the arrangement of CDRs is somewhat dif-
ferent in the camel and shark heavy chain variable regions,
they share many characteristics, including extremely high
stability. They remain functional after 100?C heat and ex-
treme pH treatment.
Accumulating reports have demonstrated the therapeutic
potentials of camelid antibody-based fragments in treat-
ing cancer and neural diseases. They can even be used
in shampoo to prevent dandruff. For basic research, these
tiny antigen binders can result in quantitative pull down (im-
munoprecipitation or co-immunoprecipitation, co-IP) with
unmatched efficiency. Nanobodies can also bind to previ-
ously inaccessible protein structure clefts such as enzyme
active sites, penetrate in vivo barriers, and provide libraries
for binding partner selection.
Allele Biotech has been working on display antibody selec-
tion since its early days through an NIH grant. We carried
out a NIH/NCI contract for scFv yeast display in 2008 in col-
laboration with our partners. In 2009 Allele Biotech is intro-
ducing camelid antibody based products to the US market
by working with Chromotek.