MEASURING PROTEIN SIZE - SDS POPLYACRYLAMIDE GEL ELECTROPHORESIS

Even “simple” cells like erythrocytes or extracellular fluids like plasma contain large numbers of proteins. It is often useful to fractionate such mixtures to ask whether a particular protein is present in an abnormal amount or has unusual activity. Every protein folds into a compact three-dimensional shape determined by its amino acid sequence. These structures are organized so that nonpolar amino acid side chains mostly face inwards away from the surface of the protein, while polar side chains, especially charged ones, face outwards and interact with water molecules in the protein’s environment. As a result, proteins can differ from one another not only in their sizes, but also in their shapes, solubilities in water, and in the net charge and charge distribution on their surfaces. Fractionation schemes in clinical assays of human proteins use these distinguishing features. We will focus on one approach, fractionation by size, and one specific fractionation technology, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.

In order to separate proteins by size, the effects of shape and charge must be eliminated. As shown in the top part of the diagram, a polypeptide chain in the presence of the detergent SDS unfolds and binds approximately one SDS per every two amino acid residues. The hydrophobic tails of the SDS molecules interact with the unfolded polypeptide while the charged sulfate head groups of the SDS molecules protrude away from the polypeptide. The sulfate groups repel one another, holding the polypeptide in a rod-like conformation. When a mixture of proteins is treated in this way, every protein takes on the same rod-like conformation with a uniformly dense coating of negative charges. (The number of charges contributed by the sulfate groups is very large compared to the ones from charged side chains in almost all cases.) When a mixture of SDS-treated proteins is applied to one end of a polyacrylamide gel and an electrical charge is applied to the gel, the proteins migrate in the electrical field at rates determined by their sizes. Specifically, as shown the graph in the diagram, each protein moves at a rate proportional to the negative logarithm of its size. This mobility is a highly reproducible feature of every protein, so by comparing the mobility of a protein of interest to those of standard proteins whose sizes are known (the dashed lines in the graph), the size of a protein of interest can be calculated.

After the proteins have been separated by size in the gel, the gel can be treated with dyes that react strongly with all proteins, yielding a pattern of bands like the one shown below the graph. How can we pick out the one protein of interest in our assay, for example an enzyme that we suspect has low activity, causing our patient’s symptoms? Reagents are needed to identify specific proteins. One general strategy is the use of specific antibodies. The proteins can be blot-transferred from the gel onto a membrane, and the membrane can then be incubated with a solution of antibody (typically coupled to a reagent that makes bound antibody easy to visualize). The membrane is washed, the antibody is visualized, and the protein of interest is highlighted as indicated in the bottom pattern. The amount of bound antibody is a semiquantitative measure of the amount of protein present, so an assay like this can be used to detect under- or overproduction of a protein of interest.

What if we suspect that the protein is present in usual amounts but is not functioning? In a number of cases, assays for molecular function have been developed that can be carried out directly on the membrane. SDS is washed away; proteins are allowed to refold in place, and the assay is done. For example, to detect an enzyme, the membrane can be incubated with a colorless substrate that active enzyme converts to a colored, insoluble product, yielding a visible band whose intensity is a good measure of enzyme activity.

These same strategies can be applied to tissue sections (immunofluoresence and histochemistry). Also, the size fractionation strategy described here for proteins is closely related to the standard one used to separate DNA fragments for assay purposes.