Polyacrylamide Gel Electrophoresis Dr. Nikhat Siddiqi

The most widely-used polysaccharide gel matrix nowadays is that formed with agarose. This is a polymer composed of a repeating disaccharide unit called agarobiose which consists of galactose and 3, 6-anhydrogalactose. Agarose gives a more uniform degree of porosity than starch and this may be varied by altering the starting concentration of the suspension (low concentrations give large pores while high concentrations give smaller pores). This gel has found widespread use especially in the separation of DNA molecules (although it may also be used in some electrophoretic procedures involving protein samples such as immunoelectrophoresis. Dr. Nikhat Siddiqi

Polyacrylamide gels, formed by the polymerization of acrylamide and cross-linked by methylenebisacrylamide, are choice supporting media for electrophoresis because they are chemically inert and are readily formed. Dr. Nikhat Siddiqi

A far stronger gel suitable for electrophoretic separation of both proteins and nucleic acids may be formed by the polymerization of acrylamide. The inclusion of a small amount of acrylamide crosslinked by a methylene bridge (N,N methylene bisacrylamide) allows formation of a crosslinked gel with a highly-controlled porosity which is also mechanically strong and chemically inert. For separation of proteins, the ratio of acrylamide: N,N methylene bisacrylamide is usually 40 : 1 while for DNA separation it is 19 : 1. Such gels are suitable for high resolution separation of DNA and proteins across a large mass range . A wider range of molecular mass in an individual gel is achievable by the use of gradient gels. These consist of a gradient of polyacrylamide (e.g. 5–20%). Dr. Nikhat Siddiqi

Polymerisation of acrylamide to form polyacrylamide gel. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi

Polyacrylamide Nondenaturing Electrophoresis The gel network formed by polyacrylamide is a suitable environment for the electrophoretic separation of proteins in their native state, that is under nondenaturing conditions. Dr. Nikhat Siddiqi

DENATURING ELECTROPHORESIS Such experiments are especially useful in the study of proteins since they have a more varied range of tertiary structure than nucleic acids. Any biological activity or quaternary structure associated with the sample components is lost in denaturing electrophoresis. However, important structural information can be obtained. Dr. Nikhat Siddiqi

SDS Polyacrylamide Gel Electrophoresis The detergent sodium dodecyl sulphate (SDS) consists of a hydrophobic 12-carbon chain and a polar sulfated head. The hydrophobic chain can intercalate into hydrophobic parts of the protein by detergent action, disrupting its compact folded structure. SDS coats proteins with a uniform ‘layer’ of negative charges which causes them to migrate towards the anode when placed in an electrical field, regardless of the net intrinsic charge of the uncomplexed proteins. The negative charge gives a charge density largely independent of the primary structure or mass of the polypeptide. Dr. Nikhat Siddiqi

Proteins can be separated largely on the basis of mass by electrophoresis in a polyacrylamide gel under denaturing conditions. Mercaptoethanol (2-thioethanol) or dithiothreitol also is added to reduce disulfide bonds. Dr. Nikhat Siddiqi

The SDS-protein complexes are then subjected to electrophoresis. When the electrophoresis is complete, the proteins in the gel can be visualized by staining them with silver or a dye such as Coomassie blue, which reveals a series of bands. Radioactive labels can be detected by placing a sheet of x-ray film over the gel, a procedure called autoradiography. Dr. Nikhat Siddiqi

Staining of Proteins After Electrophoresis Proteins subjected to electrophoresis on an SDS-polyacrylamide gel can be visualized by staining with Coomassie blue. Dr. Nikhat Siddiqi

Although most proteins stain with this dye, it should be noted that not all proteins take up the dye with equal affinity. Less sensitive protein dyes include ponceau red and amido black. Ponceau red has the advantage that it stains reversibly and may be removed from the protein to allow subsequent analysis. The most sensitive staining method for protein is silver staining. This involves soaking the gel in AgNO3 which results in precipitation of metallic silver (Ag0) at the location of protein. Radioactive proteins/nucleic acids can be visualized after electrophoretic separation by autoradiography. Dr. Nikhat Siddiqi

Electrophoresis Can Determine Mass The electrophoretic mobility of many proteins in SDS-polyacrylamide gels is inversely proportional to the logarithm of their mass. (K. Weber and M. Osborn). Dr. Nikhat Siddiqi

Discontinuous polyacrylamide gel electrophoresis Dr. Nikhat Siddiqi

Stacking gel has a low concentration of polyacrylamide (3–5%), low ionic strength and a pH near neutrality. The resolving gel, by contrast, is composed of a higher percentage of acrylamide (8–20%), higher ionic strength and an alkaline pH. This gel achieves separation of sample molecules stacked at the interface. The lower ionic strength of the stacking gel causes higher electrical resistance and hence stronger electrical field strength in this gel compared to the resolving gel. This means that, at a given voltage, samples have higher mobility in the stacking compared to the resolving gel. Dr. Nikhat Siddiqi

Isoelectric Focussing Suppose that a mixture of proteins undergoes electrophoresis in a pH gradient in a gel in the absence of SDS. Each protein will move until it reaches a position in the gel at which the pH is equal to the pI of the protein. This method of separating proteins according to their isoelectric point is called isoelectric focusing. The pH gradient in the gel is formed first by subjecting a mixture of polyampholytes (small multicharged polymers) having many pI values to electrophoresis. Isoelectric focusing can readily resolve proteins that differ in pI by as little as 0.01, which means that proteins differing by one net charge can be separated. Dr. Nikhat Siddiqi

Isoelectric Focusing A pH gradient is established in a gel before loading the sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net charge. (B) The proteins form bands that can be excised and used for further experimentation. Dr. Nikhat Siddiqi

Dr. Nikhat Siddiqi