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Study Notes: Denaturing Gels - Proteins

This section deals mainly with SDS-PAGE and you need to be aware of factors affecting the use of denaturing gels. In their native form proteins fold into a variety of shapes such as compact (globular) and elongated (Alpha -helical), into sheets (Beta -pleated) or are even composed of protein subunits. In their native form migration through a gel would reflect their relative shape (globular proteins would migrate faster) rather than their actual molecular weight. Denaturing the protein overcomes the anomalies related to the protein shape and masses determined by SDS-PAGE are usually accurate within 5 - 10%.

Remember that molecular weight is actually a ratio and is called the relative molecular mass and denoted by the symbol ‘Mr’.

Some factors affecting SDS-PAGE

Denaturant
The most commonly used denaturant is SDS (sodium dodecyl sulfate), which is a surfactant (detergent). It denatures proteins by binding to the protein chain and effectively coating the protein and does so in a uniform manner (1.4 g SDS/g protein). SDS carries a negative charge and hence imparts a total charge to the protein that is directly related to the length (size) of the protein. This means that in SDS-PAGE, separation of the proteins occurs by mass alone. The smaller the protein the faster the migration and vice versa.

The detergent, SDS, imparts a negative charge when it attaches to proteins. The size of the negative charge is directly proportional to the size of the protein.

Sample preparation
SDS is a great detergent and will solubilise many cells and tissues making sample preparation for SDS-PAGE relatively easy. Samples are heated at 95 - 100°C to completely dissolve them and sometimes a stronger sample buffer is used containing more SDS and DTT (dithiothreitol - a reducing agent to break down disulfide bonds in the protein).

The goals of sample preparation are to:

  • fully denature the proteins
  • disrupt any disulfide bonds
  • dissolve any particles in the sample.

A common sample buffer would be:

  • 125 mM Tris-HCl, pH 6.8
  • 4% SDS
  • 5% w/v DTT
  • 10 mg/mL Bromophenol Blue
  • 25% v/v Glycerol.

The Bromophenol Blue is used to colour the sample and the glycerol is to make the sample heavy so that is will sink to the bottom of the sample well during loading and not float up and be lost.

The gel
Gels may be continuous or discontinuous but in SDS-PAGE a discontinuous gel system is used. The Laemmli gel has an upper ‘stacking gel’ of low percentage (3% acrylamide) with a low pH (6.8) and a lower ‘separating gel’ of higher percentage (say 5% - 20% depending on requirements) and higher pH (8.8). Both only contain chloride as the mobile anion.

Tank buffer
Upper and lower tank buffers have glycine as the mobile anion and are at a pH of 8.8.

Pouring the gel
Pouring the gel is done in two stages. The bottom separating gel is poured first and allowed to polymerise under a layer of water or butanol to ensure an absolutely flat top surface. The water is then removed, the stacking gel poured in and a sample comb inserted in the top of the gel. The sample comb forms the sample wells and is carefully removed after polymerisation and prior to use. Both gels are poured carefully to avoid bubbles and the apparatus is carefully assembled to avoid leaks. Both bubbles and leaks will affect the electrophoretic run and lead to artefacts in the samples.

 

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