炳洋科技-技术服务

技术服务

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:

::	Straight and continuous pH gradients, dpH/dx.
::	Constant field strength, E
::	The two different proteins have the same diffusion coefficient, D.
::	The electrophoretic mobility change with pH, dμ /dpH, is constant and the same for both proteins (This is not a good assumption).
::	Two closely spaced proteins are considered are considered separated when the position of their peak maxima differs by 3 standard deviation or more.

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

Equations 2, 3, 4

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.
The higher the pH dependence of the mobility, (dμ /dpH), the better the focusing. A high electrophoretic mobility close to the IpH will efficiently transfer diffused protein molecule back to IpH. This is essentially an intrinsic factor for the protein that cannot be manipulated. The effect of modifying mobility by affecting the viscosity will be counteracted by the effect of viscosity on diffusion as already discussed and the overall effect is difficult to predict. A high value of dμ /dpH results from the presence of many groups with pKa values close to the IpH. Statistically this is more likely to be the case for a larger than for smaller proteins. Both dμ /dpH and diffusion thus favor the focusing of large proteins and the influence of these factors explains the difficulties of focusing small proteins and peptides to sharp zones.

Figure 2: Principle of isoelectric focusing.

Figure 2 is a schematic illustration of a sample with two proteins P1 and P2 place in the center of a pH gradient. P1 is positively charged and will migrate toward the cathode; P2 is negatively charged and will migrate toward the anode. As the proteins approach their IpH, they gradually become less and less charged. The proteins will thus concentrate at the position where pH = IpH. The proteins cannot concentrate in a indefinitely concentrated zone. widening by diffusion is inevitable. Any protein molecule diffusing away from the IpH will acquire a net charge and be transferred back to IpH again by electrophoresis. A balance will be set up between electrophoretic accumulation at IpH and diffusion.

Formation of Natural pH gradients
The formation of a pH gradient is schematically illustrated in Figure 3. Hydrogen ions form at the anode and hydroxyl ions at the cathode in the electrode reactions. This results in region of low and high pH near the anode and cathode respectively and steep pH gradients as one moves into the bulk solution. An amphoteric species with a IpH lower than the average pH in the system will concentrate in the steep gradient close to the anode. A substance with good buffering capacity at its IpH will create a H plateau around its IpH. Given a sufficient number of such substances with evenly distribute IpH values their corresponding plateau will overlap, resulting in a continuous pH gradient. The amphoteric substances that form and stabilize the pH gradient are collectively called carrier ampholytes.

The most essential property for a good carrier ampholyte molecule is a good buffering capacity at its isoelectric point. This requires many pK values close to the buffering capacity at its isoelctric point for each molecular species, making most naturally occurring ampholytic substance, especially most naturally occurring amino acids, useless as carrier ampholytes. Svensson realized that the only way to produce suitable carrier ampholytes was to synthesize substances with the required properties. It was not until the first synthetic carrier ampholytes were successfully prepared that isoelectric focusing could be developed into the practically useful technique of today.

The established pH gradient is maintained by hundreds or thousands of carrier ampholytes molecules lined up in order of IpH with partially overlapping distributions. Since there are no other ionic species in the system, each carrier ampholyte must act as counter ion to other carrier ampholytes consequently each position in the pH gradient will have a unique chemical composition. Electrical conductance and buffer capacity will therefore vary over the pH gradient. Regions with low buffer capacity are more prone to distortion. In preparative experiments with protein loads, buffering capacity form the proteins may affect the pH gradient.

Local heating will occur in the regions with the highest field strength (lowest conductance) and these regions will determine how high an overall voltage can be used. Consequently, other regions with lower field strength will no be focused at optimal conditions. Optimal conditions over the whole pH gradient thus requires even field strength conductance and buffering capacity across the gradient.

A large number of carrier ampholyte mixture are available giving different pH gradients. Many can also be obtained in precast gels ready to use. The optimal pH gradient will depend on the purpose of the experiment. For screening purposes, a broad range interval (pH 3-10 or similar) should be used. A narrow pH range interval is useful for careful IpH determinations or when analyzing proteins with very similar IpH points. Generally, one should not use a narrower gradient than necessary because the shallower gradient will lead to longer focusing times and more diffuse bands. When choosing pH gradient one should be aware that the interval stated by the manufacturer can only be an approximation. The exact gradient obtained depends on many factors such as choice of electrolyte solutions, gradient medium (PAA or agarose), focusing time etc.

Despite the large number of pH intervals available, there may be occasions where none of them fits perfectly. In such cases one can either choose to work with Immobiline or use "pH gradient engineering" in any of the following variants:

::	Extend a given pH interval by adding carrier ampholytes covering the adjacent or a partly overlapping region. Extension into the extreme pH ends can be accomplished by adding acidic or basic compounds.
::	Expand a certain pH area by adding an amphoteric substance, "spacer", such as an amino acid. The spacer should be a "bad" ampholyte so that it does not focus too well.
::	Extend a certain pH range by manipulating the thickness of the gel. The gradient will be shallower in areas with thinner gel.
::	Manipulating the carrier ampholyte concentration will also affect the steepness of the final gradient. Areas with lower concentration will give shallower gradients.

The different methods can be combined, as was demonstrated by Gill.

Generally IEF will give a true representation of the isoelectric spectrum of the sample. However, IEF of immunoglobulins in standard carrier ampholyte mixtures results in distinct bands in the otherwise continuous smear of immunoglobulin molecules. This was shown to depend on heterogeneity in the carrier ampholyte distribution. For a more truthful representation of the distribution of IpH points in an immunoglobulin sample, the best results were obtained in mixtures of different carrier ampholyte preparations. A mixture of three different Pharmalyte intervals to maximize the number of carrier ampholytes in the interesting region was found to give the best results.