Tuesday, 21 October 2014

Understanding the SDS PAGE results

The first batch of gels were stained and de-stained this week, revealing some interesting observations and some frustrations. The first thing to say is that the gel on the left reveals that the insoluble fraction (i.e. the proteins precipitated by the addition of an equal volume of 80% (w/v) saturated ammonium sulphate led to considerable enrichment of a low molecular weight protein (the blue blob at the foot of the gel). There are between 3-4 visible bands starting from the point where the stacking gel meets the resolving gel. The latter interface is a point where very high molecular weight proteins or often precipitates collect and these should be ignored here. This leaves a feint band followed by a significant band, approximately half way down the gel. The three bands illustrate that proteins of quite different levels of abundance are precipitated by the ammonium sulphate and that they differ considerably with respect to molecular weight. The blob is the lowest molecular weight of the three protein species. So we have quite a lot of information from this first gel. However, there are no molecular weight markers (from which we could deduce the molecular weight of the three bands) and the gel itself isn't a triumph of lab technique! Let's look at the problems.

First the gel samples contain a relatively high concentration of salt (you added an equal volume of approximately 5M ammonium sulphate to your protein sample: calculate the salt concentration in the sample you loaded onto the gel). Secondly, this was the first time you have loaded samples onto a gel of this type and it is a little tricky (see the middle lanes of the same gel (RHS). Sometimes samples flow over into adjacent lanes. Nevertheless, despite these issues, which can all be overcome with practice, we can see that the value of ammonium sulphate as a simple method for the enrichment of proteins. In fact whilst it is used to achieve some level of purification, perhaps its greatest value it brings, is in concentrating proteins, which can often help stabilise proteins and keep sample volumes down to manageable sizes. What simple technique could you employ to remove the ammonium sulphate from the sample before applying to the SDS PAGE? 

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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