Ch 5: Protein Purification and Characterization Techniques

Extracting Cellular Protein: Rupturing Cells

 Know the general steps of extracting cellular proteins
o Isolate target protein from the hundreds of proteins present in a single cell. This includes eliminating contaminants
to obtain a pure sample.
 In order to access the cell, cell rupture must occur.
 Know purpose of this step
 The first step: Homogenization is a common technique for breaking cells open; there are many options –
 May use a blender with a suitable buffer but it also breaks subcellular organelles
 Cell rupture can be induced using a Potter-Elvehjem homogenizer – a gentler approach
o It contains a thick-walled tube through which a tight fitting plunger is passed.
 The squeezing of the homogenate around the plunger breaks open the cell
membrane while leaving many organelles intact.
o The mixture is strained to remove extraneous tissue and cellular structures
 The proteins are fully soluble in the liquid fraction.
o Used to maintain the structural integrity of the subcellular organelles
 Tissue must be soft enough for use: liver
 Sonication – uses sound waves to break open the cell
 Freezing, thawing
o Next, ruptured cells can then be subjected to differential centrifugation several times
 If the protein of interest is solidly attached to a membrane, detergents need to be added to detach it.
 Know the general steps of differential centrifugation
 The sample is spun at high speeds to precipitate solids – centrifuged – and the force of gravity is
increased each time to extract smaller and smaller cell components in the pellet.
o With each increase in force, various cellular components are extracted.
 Differential speeds allow for particle separation.
o Once the cellular extract is separated from the membrane fraction and collected, protein purification begins
 Purification – once cellular content is released, purify the soluble protein of interest based on its solubility
o Isolating a soluble cytosolic protein using one spin
 After disrupting the cell membrane, soluble cytosolic proteins will be free of the cells.
 Spinning at 100,000 × g will pull down the cell microsomal fraction and debris, leaving soluble
cytosolic proteins in the supernatant.
o Further purification will be needed to separate the target protein from other soluble
cytosolic proteins.

Review Exercises Recommended: 2

Extracting Cellular Protein: Protein Purification

 The early stages in protein purification often involve methods with broader applications and intended to remove significant
amount of contaminating proteins – salting out is a method used.
 Know the general principles of salt fractionation (salting out)
o Based on the differences in solubility of proteins in salt solutions
 Protein solubility relates to its net charge, ionic strength of the solution, and polarity of the protein.
 Most common salt used: ammonium sulfate (NH4SO4)
o Preferred b/c a high salt concentration may be achieved with little change in solution
density – advantage of centrifugation.
 Lets you add a large amount of salt in a small volume of solution
 This is critical to preventing the over-dilution of your solution
 Amino acid content and arrangement make some amino acids more soluble than others
o A protein with more highly polar amino acids on the surface is more soluble than those
with more hydrophobic ones.
o Salting out is a process whereby a highly ionic salt is used to reduce the solubility of a protein until it comes out of
solution and can be centrifuged.
 The salt forms ion–dipole bonds with the water in the solution, which leaves less water available to
hydrate the protein.
 The proteins are forced to interact with each other, and small hydrophobic patches on the proteins
associate with each other. These local aggregations build such that large aggregates, which are not
soluble, form, and the protein aggregates precipitate out of solution.
 Nonpolar side chains begin to interact between protein molecules, and they become insoluble.
o What presence of salt ion accomplishes – draws water molecules from the solvation sphere of the protein
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 Ch 5: Protein Purification and Characterization Techniques
 When NH4SO4 is added, some water is taken away from the protein to make ion-dipole bonds with the
salts
 W/ less water available to hydrate the proteins, the proteins begin interacting w/ each other via
hydrophobic interactions
 Salt ions attract water away from the protein due to > charge density, which causes proteins to aggregate
 When enough salt (NH4SO4) is added, protein precipitates without denaturing
 Proteins with greater overall hydrophobicity precipitate first b/c they are the least soluble.
 W/ enough NH4SO4, a precipitate that contains contaminating proteins form and is centrifuged
out.
o Goal of salting out – perform multiple salt fractionations w/ increasing salt concentration to eliminate contaminating
proteins
 With each application of salt, the sample is centrifuged at high speed to pellet the precipitate into the
bottom of the tube
 Fluid supernatant – proteins that are still soluble.
 Different aliquots taken as function of [salt]
o Protein precipitates in native conformation
o Purpose of this method – prepare crude homogenate for the more procedures that follow.
 Know the general order of protein purification steps
1. Crude Extract – represents soluble fraction after cell rupture; salt fractionation
2. Salt precipitate
3. Ion-exchange chromatography
4. Molecular sieve chromatography
5. Immunoaffinity chromatography
 Know the difference between total activity and specific activity
o Total activity – total amount of target protein in the sample
 With each step, the total amount of protein in milligrams decreases as more of the contaminating proteins
are removed.
o Specific activity – total activity/total amount of protein
 Proportion of target protein compared to all present protein – a measure of protein purity
 With each step, the purity of the protein increases
o Know how these change with each step in the purification
 As the specific activity increases – that is, as we gain a higher degree of sample purity – the price you pay
is in the amount you can actually recover from the starting sample.
 High specific activity = low percent recovery.

Review Exercises Recommended: 2, 3, 4

Column Chromatography: Size Exclusion

 Chromatography – based on the fact that different compounds can distribute themselves to varying extents between different
phases or separable portions of matter.
 Know the two phases of chromatography and their purpose
o Stationary phase- selectively retards the flow of the sample, effecting separation
o Mobile phase – soluble/liquid portion that migrates through the stationary phase
 Compounds to be separated are partitioned in each phase to different degrees for separation.
 Mobile phase flows over stationary material and carries the sample to be separated with it.
 The extent of interaction of mobile and stationary phases vary b/w components
o Strong interactions are carried along more slowly than less strong ones.
 Know the method of column chromatography
o Stationary phase packed in column –
 The sample is a small volume of concentrated solution applied to the top of the column.
o Mobile phase, called the eluent, is passed through the column.
 The sample is diluted by the eluent; the separation process increases the V occupied by the sample.
 Know the method of size exclusion (gel filtration) chromatography
o It is a form of stationary phase that consists of cross-linked gel particles
 The gel particles are usually in bead form, consisting of one of two kinds of polymers: agarose or
polyacrylamide
 The cross-linked structure of these polymers produce pores in the material.
o The extent of cross linking can be controlled to select a desired pore size
o How particles separate based on size or molecular weight
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 Ch 5: Protein Purification and Characterization Techniques
 Smaller molecules can enter pores of gel, which delays their progress down the column.
 Larger molecules cannot enter the pores, and are thus eluted first; move faster down the column
o Samples are measured by UV absorption
 Gel particles can be made from two kinds of polymers via size exclusion–
o Acrylamide or agarose
 Each gel particle has a certain pore size – molecules larger than that size are completely excluded
 They remain in the mobile phase and migrate to the bottom faster.
 Smaller molecules are partially or fully included in pores – migrate more slowly.
 Sometimes in stationary and sometime in mobile phase: slower.
 The basis of separation for the following techniques
o Gel-filtration chromatography – size
 Order of elution: largest protein elutes first while the smallest elutes last
 Larger proteins travel a shorter distance b/c unlike the smaller proteins, they cannot enter the gel
bead.
o Affinity chromatography – specific ligand binding ability
o Ion-exchange chromatography – net charge
 Two elution methods:
 Raising salt concentration (primary) – salt is cheap but not as specific to a particular protein
 Changing pH – more specific for a right pI range but extreme pH can denature a protein
o Proteins become insoluble at isoelectric point.
o Reverse phase HPLC – polarity

Review Exercises Recommended: 11, 12, 19, 20

Column Chromatography: Affinity and Ion Exchange

 Know the general principles of affinity chromatography
o Based on specificity of binding to ligand
 A form of column chromatography w/ a polymeric material used in stationary phase in which the polymer
is covalently linked to a ligand, which binds specifically to a desired protein
 Have an affinity for the structural or chemical features.
 Irrelevant proteins are eluted w/ buffer while bound (substrate) protein remains in the column.
o Convenient separation method that can produce a very pure sample in one or few steps
 Know the general principles of ion exchange chromatography – molecules separated based on net charge
o Know the difference between anion and cation exchangers
 Cation exchangers – resin w/ net negative charge that binds to a (+) charged molecule flowing through
 Anion exchanger – resin w/ net positive charge that bind (-) charged molecules flowing through
o Know how pH ensures protein carries set charge and why
 Initial pH ensures protein of interest carries charge opposite to resin
 Based on pI of protein – pI is the pH in which the protein has no net charge
 W/ appropriate pH, protein binds to resin
o Know which molecules flow through
 Proteins with same charge as resin and other neutral proteins flow through – mobile phase
o Know how protein eluted from column
 The exchange resin is bound to counter ions
 Proteins with a net charge opposite to that of the exchanger remain in the column, exchanging
places with the bound counter ion
o When the salt concentration increases, the bound proteins are outcompeted by salt ions.
 Changing the pH alters the net charge of the bound protein. At its pI, the protein
will elute b/c it has no net charge.
 Displace protein from the column by adding high amounts of salt inducing a competitive
displacement of charges. Ions bind the resin and displace protein
o Know how to determine order of elution of components, based on charge
 Molecules elute based on charge
 Amino acid with the highest opposite charge will bind more strongly to resin, thus eluting last.
 Molecules are eluted based on changes in pH or raising salt concentration
 In a cationic exchange: aspartate does not bind thus it elutes first followed by the net neutral charge.
 The column resin has a negative charge and aspartate is negative.
 Lysine elutes last due to its positive basic charge.

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 Ch 5: Protein Purification and Characterization Techniques
 Know general purpose of HPLC – high performance liquid chromatography
o Fast and clean purifications that separates based on polarity
o Reverse phase HPLC – nonpolar stationary phase and polar mobile phase
 Used for separating nonpolar molecules
 High resolution columns and takes less time to separate.
 Design an experiment to purify protein X on an anion-exchange column. Protein X has an isoelectric point of 7.0.
o Set up an anion-exchange column, such as Q-sepharose (quaternary amine).
o Run the column at pH , a pH at which the protein X has a net negative charge.
o Put a homogenate containing protein X on the column and wash with the starting buffer.
o Protein X will bind to the column. Then elute by running a salt gradient.
 An amino acid mixture consisting of lysine, leucine, and glutamic acid is to be separated by ion-exchange chromatography,
using a cation-exchange resin at pH , with the eluting buffer at the same pH. Which of these amino acids will be eluted from
the column first? Will any other treatment be needed to elute one of these amino acids from the column?
o Glutamic acid will be eluted first because the column pH is close to its pI. Leucine and lysine will be positively
charged and will stick to the column. To elute leucine, raise the pH to around 6. To elute lysine, raise the pH to
around 11.

 An amino acid mixture consisting of phenylalanine, glycine, and glutamic acid is to be separated by HPLC. The stationary
phase is aqueous and the mobile phase is a solvent less polar than water. Which of these amino acids will move the fastest?
Which one will move the slowest?
o A nonpolar mobile solvent will move the nonpolar amino acids fastest, so phenylalanine will be the first to elute,
followed by glycine and then glutamic acid

 In reverse-phase HPLC, the stationary phase is nonpolar and the mobile phase is a polar solvent at neutral pH. Which of the
three amino acids – F, G, E – will move fastest on a reverse-phase HPLC column? Which one will move the slowest?
o The nonpolar amino acids will stick the most to the stationary phase, so glutamic acid will move the fastest,
followed by glycine and then phenylalanine.

Review Exercises Recommended: 14, 15, 23, 27, 28, 29

Electrophoresis
 Know general principles of separation by electrophoresis
o Electrophoresis – motion of charged particles in an electric field towards an electrode of opposite charge.
 Macromolecules have different mobilities based on their charge, shape, and size.
 An electric current is passed through the medium at a controlled voltage to achieve the desired separation
o Separate based on charge to mass ratio
 Mobilities differ based on charge, shape, size of molecules
o Agarose – used mainly to separate nucleic acids and may be used for native gel separation of proteins
o Polyacrylamide – usual gel for protein separation
 More resistance towards larger molecules
 Know the methods of SDS-PAGE – sodium dodecyl sulfate
o Purpose of SDS – separates proteins solely on size or molecular weight
 SDS completely denatures proteins breaking all noncovalent interactions that determine secondary,
tertiary, and quaternary structures
 Allows all proteins to have roughly the same shape – a random coil – such that they can migrate in the gel
solely based on their native size.
 The negative charge on SDS imparts a net negative charge on the protein
o How size relates to mobility (smaller is faster)
 Protein is coated with SDS, which denatures the protein and coats it w/ negative charge
 - charge causes it to move towards the + electrode
 Mobility is inversely related to size on a log scale
 Smaller molecules move faster.
 Know method of isoelectric focusing – variation of electrophoresis
o Substances separated based on pI – positive and negative charges are equal
 Proteins differ in their titratable groups thereby differing in isoelectric points (pI).
o Gradient used and how separation accomplished
 Gel is prepared w/ a pH gradient increasing up the gel.
 Proteins migrate down the gel under influence of electric field so long as they are charged
 Once the protein reaches a pH equal to its pI, it becomes neutral and will STOP moving.

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 Ch 5: Protein Purification and Characterization Techniques
o Each protein’s position on the gel corresponds with its pI
 General method of 2D electrophoresis – combining techniques
o What are the two dimensions
 1st – sample applied to isoelectric focusing gel
 Proteins separated based on charge
 2nd – SDS-PAGE at 90 degree angle to separate samples by size
o What is the purpose – better separation of complex mixtures.

Review Exercises Recommended: 31, 35, 37, 38, 39*

*Q39: The solution to this problem is wrong – though I like the question. If you look at the image – you can plainly see that the
protein size cannot be more than 36,000. If you use Excel to plot distance traveled by the log of the MW – you should get something
on the order of 32,400 Daltons.

Primary Structure Determination: Edman Sequencing

 Know purpose for producing various mixtures of peptides
o Trypsin cleaves peptide bonds at the site of a positively charged R group – K, R, H
 The cleavage takes place in such a way that the amino acid with the charged side chain ends up on the C-
terminal site, despite the location it held on the polypeptide.
o Chymotrypsin – cleaves peptide bonds at the site of aromatic amino acids – tyrosine, tryptophan, phenylalanine
 They end up at the C-terminal end of the peptide
o Cyanogen bromide (CNBr) – sites of cleavage are at internal methionine residues
 Methionine’s Sulfur reacts w/ the Carbon of CNBr to produce homoserine lactone at the C terminal end
o The cleavage of proteins by these reagents produces a mixture of peptides, which are then separated by high
performance liquid chromatography
 The use of several reagents on different samples of a protein to be sequenced produces different mixtures
 The sequence of a set of peptides produced by one reagent overlap the sequences produced by
another reagent.
o As a result, the peptides can be arranged in the porper order aftehr their own sequences
have been determined.
 Know the benefits and limitations of Edman sequencing
o Edman degradation – a method for determining the amino acid sequence/order of peptides and proteins by cleaving
each amino acid in the sequence.
 Sequence of peptide containing 10-40 amino acids determined in 30 min
 Limitation – the method becomes more difficult as the number of amino acids increases: > 100 residues
 Its necessary to break a long polypeptide into fragments
 The signal degrades overtime
 Benefits – efficient and fast in determining N and C terminals
 The overlapping sequences of the peptides produced by different reagents is key to solving
primary structure.
 Each round leaves the rest of the amino acid in tact
 Know the general steps
o The Edman reagent, PITC or Phenyl isothiocyanate, reacts with the peptide’s N-terminal residue under mildly
alkaline conditions
 Deprotonated and unmodified N acts a nucleophile and attacks of carbonyl on PITC
 The modified amino acid can be cleaved leaving the rest of the peptide in tact
 It can be detected as the phenylthiohydantoin derivative of the amino acid
o TFA (trilfluoroacetic acid)’s anhydrous treatment hydrolyzes the peptide bond and releases terminal amino acid
 This cyclizes the structure to release the N-terminal amino acid residue
 It can be detected as a
thiazolinone derivative
o Organic extraction and treatment w/ aqueous
acid yield the N-terminal amino acid as
phenylthiohydantoin (PTH)
o A “sequencer” is used to repeat the process on
each amino acid of the polypeptide.

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 Ch 5: Protein Purification and Characterization Techniques
Protein Detection Techniques
 Know general method and purpose of mass spectrometry
o Mass spectrometry – uses differences in mass-to-charge ratio to separate and identify molecules in primary structure
 Most common method: electrospray ionization – solution of molecules sprayed from capillary/opening
under a strong electric field. Particles become ionized/charged
 Differently charged species give a different series of charge to mass peak
o Accurate technique
 Know general steps and purpose of ELISA [Page 125] – enzyme linked immunosorbent assay on a microtiter plate
o ELISA is based on reactions between proteins and antibodies
 Antibodies bind specifically to proteins that elicited their creation
 They make for excellent tag markers that seek out and bind to their target protein.
o Purpose of various components -
o Protein is detected using two different antibodies:
 Primary antibody (Ab) – binds specifically to target protein
 Secondary antibody – binds to primary antibody apoach in order to locate the primary antibody.
 It contains a marker, which is an enzyme that catalyzes a specific reaction
 The enzyme link: secondary antibody is covalently attacked to the primary
o When the enzyme substrate is added, it generates a detectable product: color or
fluorescence
 Know the general methods associated with protein chips
o Protein microarray – small plates of only a few centimeters that may contain tens of thousands of implanted proteins
 ELISA + western blot + power of tremendous throughput
o How a protein detected – accomplished via fluorescence
 The relative colors and intensities tell the researcher which proteins are found in the various spots and how
much of each is present.
 Know the general principles of the Western blot
o Why protein transferred to nitrocellulose
 Protein separated by gel electrophoresis, usually SDS-page, then transferred to nitrocellulose membrane
 Its transferred to subject the protein for further analysis and identification using methods not
applicable in a gel environment.
o How detection accomplished:
 Transfer from gel to membrane is accomplished by creating a paper sandwich of sorts soaked w/ buffer
 The membrane is placed under the gel and a current is applied
o The (-) charged protein moves towards the + charged anode, thereby leaving the gel to
deposit on the nitrocellulose membrane.
 Detection on the membrane is similar to ELISA – uses primary and secondary
antibodies.
Review Exercises Recommended: 56
Proteomics
 Know what constitutes a proteome
o Proteomics – systematic analysis of an organism’s complete complement of proteins, its proteome.
 A given cell produces several thousands of proteins at a given time: difficult to study.
o Subdivisions: structural, expression, and interaction
 Know the purpose and types of studies
 Know the general steps of using protein bait to analyze protein interactions
o “Bait” proteins are created and allowed to. Bind to an affinity column.
 In binding the column, they take other bound proteins with them. The bound complex is eluted from the
column, purified with SDS-page
 The bands are excised from the gel and digested with trypsin.
o After digestion, the pieces are identified with mass spectrometry.
o What is the purpose of the tag on the bait protein?
 The bait protein is constructed to have a specific affinity tag. The bait protein interacts with cell proteins of
interest and then binds to an affinity column via the tag
 In this way,m cell proteins of interest can be found and isolated.

Review Exercises Recommended: 64

I. Order of steps for enzyme isolation

a. Homogenization
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 Ch 5: Protein Purification and Characterization Techniques
b. Salting out
c. Column Chromatography
d. Electrophoresis
II. 31) What physical parameters of a protein control its migration on electrophoresis
a. Size, shape, and charge
III. 35) In a mixture of proteins with differing sizes, shapes, and charges separated by electrophoresis, which proteins move
fastest towards the positive electrode/anode?
a. Proteins w/ highest charge/mass ratio move the fastest
IV. 37) How does adding SDS to proteins affect the basis of separation on electrophoresis
a. SDS binds to the protein in a constant ratio of 1.4 SDS per gram of protein
i. It coats the protein w. negative charge and puts it in a random coil shape. Thus, charge and shape are
eliminated.
V. 39)