advances-in-protein-chemistry

» Denaturation of all proteins gives them a similar rod-like shape.
This allows the movement of all the proteins towards positive electrode at a rate that depends only on the molecular size of the protein.
The smaller proteins experience a lower degree of sieving in the get and move faster as compared to larger proteins. In SDS-PAGE, it is
common to run proteins of known molecular weight (molecular weight markers) in a separate lane in the gel, in order to calibrate the gel
and determine the approximate molecular mass of unknown proteins by comparing the distance travelled relative to the marker.
Apart from molecular weight determination, SDS-PAGE may also be effectively used for the checking the purity of a protein sample.
It may also be a useful technique for the separation and purification of proteins, when obtaining a protein in its native form is not
essential (such as for immunization. Automated SDS-PAGE systems are also available for preparative purposes (Figure 9C).
Figure 9: A. A verticl gel electrophoresis assembly (power supply not shown). B. A gel showing proteins bands after staining with Commassie
Brilliant Blue R 250. C. A preparative SDS-PAGE assembly.
Isoelectric Focusing: The principle of separation in isoelectric focusing (IEF) is quite similar to chromatofocussing discussed in
section 1.2.2.7. However, IEF combines the use electric current with a pH gradient in the gel in order to separate proteins according to
their pI, which falls between pH 3 to pH11 for most of the proteins. Since the pI is a characteristic of a protein, IEF provides an excellent
resolution. The movement of a protein through a pH gradient leads to a change in its net charge. Under electric field, the protein migrates
to the pH value where its net charge becomes zero (that is its pI). The protein will stop at this position as due to a zero net charge it will
not experience any pull under electric field. In other words, it is focused in the region of its pI. Similarly, other proteins in the mixture
will also separate according to their pI values with a high degree of resolution.
A wide or a narrow range of pH may be chaosen for generating a gradient. A stable and continuous pH gradient between the between
the electrodes is maintained by one of the following methods:
» Carrier ampholytes; which contain a heterogeneous mixture of small molecular weight compounds that carry multiple charges of
both types, with closely spaced pI values. Chemically they are polyaminopolycarboxylate compounds. When the voltage is applied, the
ampholytes align themselves in the gel according to their pI values and tend to buffer the pH in their proximity. This establishes a stable
pH gradient.
» Immobilized pH gradients (IPG) strips; are formed by covalently linking the buffering groups to a polyacrylamide gel. A pH
gradient of a desired range is generated by a gradient of different buffering groups.
IEF can be run under either native or denaturing conditions. Native IEF retains protein structure and enzymatic activity. Denaturing
IEF is performed in the presence of high concentrations (usually 8M) of urea, which denatures the protein and dissociates the subunits,
if linked non-covalently. Denaturing IEF often offers higher resolution and may give an insight into the type of interactions between
subunits of multimeric proteins.
Two-Dimensional Gel Electrophoresis: Two-dimensional electrophoresis (2DGE) was first introduced by O’Farrell [16] and Klose
[17] in 1975. The sequential application of two different electrophoresis techniques produces a multi-dimensional separation. In the
most common 2DGE, protein samples is first separated in denaturing IEF in a tube gel or an Immobilized pH gradients gel strip to
allow the separation on the basis of pI. This gel (or strip) is then equilibrated with SDS and placed on an SDS gel in the direction that is
perpendicular to direction of separation in IEF. SDS-PAGE is then carried out in second dimension to separate the proteins according to
their molecular weight. A very high-resolution is achieved in 2DGE due to the fact that it is highly improbable for two proteins to have
same pI and molecular weight simultaneously. This method enables the separation of thousands of proteins (e.g. from cell lysate) in a
single slab gel. The resulting spots can be visualized by gel staining.
3D Gel Electrophoresis: Due to a limited throughput, 2DGE may not be very suitable for comparative protein expression studies
at large-scale. 3D gel electrophoresis (3DGE) may overcome this problem. It is relatively recent technique developed by Ventzki and
Stegemann [18] that gives a much higher resolution than 2DGE and a larger number of samples can be simultaneously analyzed. Following
conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body,
and the samples are transferred electrokinetically to the SDS gel. Since there may be unequal heat generation, special care is required here
so that SDS-PAGE occurs under identical electrophoretic and thermal conditions, in order to avoid gel-to-gel variations. The proteins
are labeled with fluorescent tag and the protein bands are resolved online by photodetection of laser-induced fluorescence (LIF). A digital
camera placed below the gel captures the images in real time as the fluorescently labeled proteins pass through the laser-illuminated
detection plane. Image processing software simultaneously processes these images, making results immediately accessible without
further gel processing [19]. The separation patterns are analyzed using special software (available commercially e.g., Kapelan LabImage
1D or Decodon Delta2D), thereby improving the comparability, resolution and efficiency of the separation. Though still in the stages of
development and improvisations, the method is set to find use in a wide range of applications in clinical diagnosis and pharmacology by
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