

Just a few technical points on the preparation and treatment of the gels. The gel is a polymer which is formed from a solution of acrylamide and bis acrylamide. When ammonium persulphate and TEMED are added to a mixture of acrylamide and bis acrylamide, the acrylamide polymerises and the bis acrylamide forms cross links. By adjusting the concentration and proportions of acrylamide and bis acrylamide, the gel forms a "cross linked net" which sieves the proteins. The gel is often made in two parts, with the lower 75% called the resolving gel (often 10% polyacrylamide) and the upper gel, called the stacking gel, often a lower percentage. The stacking gel sets a little slower and it is into this layer that we push a "comb" which is the same thickness as the plastic spacer that allows us to form the gel between the two glass plates. The comb is removed prior to loading samples (which are typically 5-50ul in volume. The gel forms a bridge between the upper and lower buffer chambers and when the buffers contain the detergent SDS, the proteins are generally electrophoresed in such a way that they run as a range of negatively charged "rods" whose length is determined by their primary structure (number of amino acids).
The sample loading buffer contains a few specific components: bromophenol blue is a dye that doesn't bind to proteins, but allows us to track the samples, ensuring that we don't run the gel for too long. The smell is due to the presence of beta mercaptoethanol, a reducing agent which ensures that those proteins containing disulphide bonds are fully unfolded. Finally glycerol is added in order to facilitate sample loading: the density of the blue protein sample making it easy to load into the wells of the gel, and easy to tell if your sample has spilled over into an adjacent lane. At the end of the electrophoresis phase, typically one hour, the clear gel is removed carefully from between the plates (as I demonstrated) and is covered by a solution of "stain". This solution contains Coomassie Brilliant Blue, dissolved in a mixture of methanol, acetic acid and water (hence the smell!). The dye stains both gel and proteins, and we have to actively destain the polyacrylamide with a similar solution (omitting the dye). The whole process of staining and destaining takes a few hours (often destaining is carried out overnight), and the gel is usually contained in a plastic box and gently agitated. In the next methods blog, I will look at the gels that you obtain after the column chromatography step.
Key words SDS PAGE, sample buffer, ammonium sulphate fractionation gel composition
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