Protein Separation

Polyacrylamide Gel electrophoresis (PAGE) - The acrylamide matrix allows smaller proteins into the pores where they can migrate through the gel. Movement of larger proteins is slowed because larger mass does not permit their entry into the pores.

Visualization of proteins is by application of dyes such as Coomassie blue (sensitivity ~1microgram per band) or if the proteins are radioactive -- by autoradiography. The latter involves placing a special film over the gel and allowing sufficient time for exposure.

Polyacrylamide Gel electrophoresis (SDS-PAGE) - Sometimes it is necessary to add SDS, an anionic detergent, to eliminate conformational differences and charge differences among/between the proteins to be resolved. With addition of SDS and beta-mercaptoethanol, proteins generally resolve according to molecular weight. Mercaptoethanol disrupts disulfide bonds and SDS intercalates into the protein backbone sufficient to impart a huge negative charge such that charge is no longer an issue. Thus proteins separate by size.

Isoelectric focusing: Proteins can also be separated based on charge, that is to say, based on the relative density of positive or negatively charged residues. The technique, when carried out on an electrophoresis apparatus, is termed isoelectrofocussing. Here as before the matrix is polyacrylamide. Polyampholytes (small multicharged polymers) are used to establish a pH gradient in the gel. Proteins are then added into the separation chamber (or well) and electrophoresed. All proteins begin migration through the matrix but halt when they have migrated to the point on the gel that reflects their pI-isoelectric point. The pI of a protein is that pH at which the protein has a net neutral charge.

Ion exchange chromatography. Another technique for separating proteins based on charge is ion exchange chromatography. Here, a column is made of resin composed of a polymer which is highly charged, diethylaminoethyl (or DEAE, positively charged) or carboxymethylcellulose (negatively charged). A buffered solution is then applied to the column together with the protein mixture. If the carboxymethylcellulose is used, then proteins with a high density of positively charged residues will bind most tightly.

After all the protein is applied, then it is followed with an increasing salt gradient (i.e., NACI). Gradually the electrostatic interaction between the protein and matrix is disrupted by the counter-ion of the salt and the protein is released and eluted. For this technique, DEAE is generally the most commonly employed and it is typically an early step in a purification protocol.

Dialysis. This technique involves separation of proteins based on size differences. Proteins are placed in a chamber or bag composed of a semi- permeable membrane. Determined by the pore size of the membrane, proteins may enter or exit the bag if they are sufficiently small to do so. At some point an equilibrium is reached and the small proteins may be removed from the system with new buffer added. Again equilibrium is reached and the small proteins can be removed. Ultimately, the larger proteins which are retained with the dialysis tubing, may be removed and studied further. Dialysis is typically a early stage procedure for protein separation

Molecular Sieve Chromatography (gel permeation chromatography)- A high resolution of proteins based on size can be obtained by this technique-arguably the most oft. used technique in biochemistry. Here, a cylindrical column made of glass is filled with a resin-generally made of a carbohydrate polymer. Like the acrylamide gel, the resin represents a 3D matrix into which proteins can migrate and attempt to work their way through the maize. Larger proteins do not enter into the beads of resin and therefore migrate more quickly through the column. They are the first to emerge at the bottom of the column. Smaller proteins enter into the beads and generally take longer to elute from the column. There are however proteins which migrate in unusual fashion, such as those which are not spherical in shape but rather more rod-like. These have difficulty in entering into the matrix and elute together with the larger proteins even though they may be quite small rods.

Salting out of proteins. The solubility of most proteins is lowered at high salt concentrations. This effect is called salting out and it is very useful. The technique is as old as the hills and generally not well understood -- if at all. Proteins respond differently to the presence of salt. Hence, this technique, when applied to a new protein, is generally accomplished only by trial and error but mostly error. Still it is everyone's first choice cause its easy. In a mixture of proteins, the concentration of ammonium sulfate is increased from ca. 0.1M to 2M and at various intervals one collects the precipitates (generally by centrifugation) and tests for the presence of the desired protein (which is either still in solution or in the precipitate). If it is still in solution, just add more of the salt, wait a while, and test the precipitate again. If you're successful, either the desired protein is precipitated selectively, or all undesirables are removed by precipitation and your wondrous protein is left standing in the salty environs of the solution --- Ah -- the stuff of fairytales. I mention this technique partly because if you happen to party with a biochemist, she or he will likely tell you stories of a successful "salt cut", in which a protein is purified in one step by this procedure. My advice: Don't listen -- its only a fairy-tale and find another party-fast!

Affinity purification. While we're on the topic of fairytales, here is the next best thing -- affinity chromatography and it really works. In this case it is important to know that most proteins function to bind something, another protein, a carbohydrate (glucose), nucleic acid (i.e., DNA or RNA), etc. If you know the "ligand" which the desired protein binds to then the ligand itself can be covalently linked to a resin. The resin is then used to fill a column and the protein mixture is applied. After many buffer washes to elute non-specifically bound material, the desired protein can be eluted by application of the ligand or by increasing the salt concentration in the buffer. I'll impart my own experiences with affinity chromatography as well as the book's rather simplistic application.

No ultracentrifugation.

Protein Analysis

Electrospray mass spectroscopy. Proteins or peptides (small proteins are called peptides) are suspended in a buffer containing a volatile acid such as trifluoroacetic acid. The solution is then injected into the instrument for analysis. The buffer and acid are quickly evaporated and the highly charged proteins remain behind. An electrical current propels the proteins much as in electrophoresis but along the path of trajectory, the proteins are deflected by a magnetic field. Big proteins could care less about the magnetic field and proceed head-on toward the detector but the smaller ones are deflected away from the center line. The degree of deflection is a measure of size and such instruments are extremely accurate - to the nearest Dalton. Accurate readings can be made up to 30 or 40,000 Daltons and this technology is advancing fast. You might ask why one needs to be so precise - considering this instrument simply an expensive toy. But in fact the power of such an instrument will help solve the mystery of what causes diseases such as multiple scleroses and rheumatoid arthritis.

Protein sequencing-The Edman Degradation. This procedure is now automated and provides for the sequential deten-nination of amino acid sequence from the amino-terminus of a protein. Generally, the technique is sensitive enough to obtain 40 to 50 amino acids of sequence. Phenyl isothiocyanate reacts with the terminal amino group of the peptide to form a phenylthiohydantoin or PTH-amino acid derivative. The identity of the PTH-amino acid is then determined by liquid chromatography (co-elution with known standards) after which another cycle is initiated. You might ask how one sequences a protein that is longer than 40 to 80 amino acids. The answer is that one must first cut your protein into pieces, sequence the pieces, and then put together the sequence puzzle.

Cleavage of proteins into peptides. This can be achieved by enzymatic or chemical means. The chemical, cyanogen bromide (CNBr), selectively cleaves proteins on the carboxy side of methionine residues. Since met residues are not too common in proteins, this technique is usually quite successful in generating a series of smaller peptides which can be sequenced. To obtain overlapping sequence information, one must however generate sequence information peptides generated by a different procedure. Thus highly specific cleavage can be obtained by trypsin which cleaves on the carboxy side of lysine and arginine residues (i.e., the basic amino acids). Chymotrypsin faithfully cleaves on the carboxyl side of the aromatic amino acids. Once both set of peptide fragments have been sequenced and overlapping sequence data is in hand, then the order of the peptides can be deduced and the sequence is known unambiguously.

Recombinant DNA technology has revolutionized how we sequence most proteins. Because of the power of the PCR technique, now it is only necessary to have a limited amount of protein sequence information to allow simple cloning of a gene. Once the gene sequence in known, the amino acid sequence can be deduced from it -- thereby sparing one of having to work with precious little protein. We will discuss PCR at a later date.

Amino acid sequences provide much valuable information.

1. Information can be used to identify the protein. i.e., is it a homologue of human hemoglobin but present in a different organism.

2. Information can be used to uncover the gene that encodes the protein. Nowadays there is an incredible DNA sequence information stored in GENBANK (derived by individuals who like to mindlessly sequence DNA and who like to party with people who discuss incredible "salt cuts"). The function of the DNA is uncovered only when one identifies a region of "BANKED" DNA sequence that may encode the protein under study. If this is to be found, then the scientist already knows the entire gene sequence ... just by computer searching. This is not uncommon and it has so transpired in my own lab!

3. Sequence information reveals repeating units or domains.

4. Proteins contain amino acid sequences which signal delivery to different regions within a cell or for excretion from the cell. Some sequences represent specific sites for cleavage and activation.

5. Sequence information can be used to construct smaller peptides which can be used to make very specific antibodies which can be used for detection or purification of the parent protein.

Proteins can be quantitated and localized by highly specific antibodies.

An antibody is a protein produced by a white blood cell in response to a foreign substance. Antibodies have specific affinity for the "antigen" (foreign substance) which elicited their production. Antibodies recognize small substances (chemicals) or small regions of a protein (an "epitope"). One can generate antibodies by injecting a mouse or rabbit with the protein (or substance) of interest several times over a month. Antibodies are then collected from blood or "monoclonal antibodies" are made from cloned white blood cells obtained from the animal. Antibodies so generated can be used to detect or localize the protein or foreign substance that was used to initiate the immune response. Use of antibodies for detection of proteins or substances by the "ELISA assay" or in the "Western blot assay". Description for generation of monoclonal antibodies is on page 366 in Stryer text.