2D Gel Electrophoresis

This is a method for the separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right angles to one another. This allows the sample to separate over a larger area, increasing the resolution of each component.

2D gel electrophoresis is generally used as a component of proteomics and is the step used for the isolation of proteins for further characterisation by mass spectroscopy. In the lab this technique is used for 2 main purposes, firstly for the large scale identification of all proteins in a sample. This is undertaken when the global protein expression of an organism or a tissue is being investigated and is best carried out on model organisms whose genomes have been fully sequenced. In this way the individual proteins can be more readily identified from the mass spectrometry data. The second use of this technique is differential expression, this is when you compare two or more samples to find differences in their protein expression. For instance, you may be looking at drugs resistence in a parasite. In this case you might like to compare a resistent organism to a susceptible one in an attempt to find the changes responsible for the resistence. Here the sequence requirements of the organism are not as important, as you are looking for a relatively small number of differences and so can devote more time to the identification of each protein.

2D electrophoresis is performed in two steps

(1) Isoelectric focusing (IEF) (First dimension electrophoresis): is used in the 1st Dimension (Righetti, P.G., 1983). This separates proteins by their charge (pI).

Isoelctric focusing (IEF) can be described as electrophoresis in a pH gradient set up between a cathode and anode with the cathode at a higher pH than the anode. Because of the amino acids in proteins, they have amphoteric propertites and will be positively charged at pH values below their IpH and negatively charged above. This means that proteins will migrate toward their IpH. Most proteins have a IpH in the range of 5 to 8.5.

Under the influence of the electrical force the pH gradient will be established by the carrier ampholytes, and the protein species migrate and focus (concentrate) at their isoelectric points. The focusing effect of the electrical force is counteracted by diffusion which is directly proportional to the protein concentration gradient in the zone. Eventually, a steady state is established where the electrokinetic transport of protein into the zone is exactly balanced by the diffusion out of the zone. From the factors that regulate the widths of the protein zones and distance between the zones, Svensson and Veterberg derived an equation for the resolution of two similar proteins, based on the following assumptions:

Equation 1: The minimum difference in IpH, for two proteins to be resolved is expressed with equation 1.

From equation 1 it can be seen that by reducing the diffusion, D, the resolution would increase. With a given separation, the only way to accomplish this is to increase the viscosity of the medium. Inert non-charged substances such as sucrose, glycerol etc. may be added or the experiment can be performed in a sieving medium such as a high concentration of polyacrylamide (PAA) gel. Increased viscosity will also affect the mobility (µ ) the mobility of the proteins. This will make the isoelectric separation longer and decrease the resolution by decreasing the dµ /dpH in equation 1_. Therefore, increasing the viscosity is not generally a successful way to improve the resolution although it may explain why there is a clear tendency for better resolution in sieving PAA gels than in more porous agarose gels.

Since the diffusion coefficient is inversely related to molecular size it follows that larger proteins will tend to focus better than smaller ones, other things being equal (See equation 2 _).

The shallower the gradient, dpH/dx (lower values of dpH/dx), the further apart will two proteins be and hence better separated. Note that the factor only applies as the square root. There are some drawbacks with use of extremely shallow gradients: Long focusing times since proteins must migrate a relatively long distance close to the IpH with very low charge: Only the limited number of proteins with IpH values within the narrow pH interval can be analyzed simultaneously; The carrier ampholine may not manage to maintain a completely smooth pH gradient. High field strength (E) will not only increase the resolution, the experimental time is also reduced. Too high field strength may give heat problems if the cooling is inefficient, especially when focusing in the very basic or acid pH region.