advances-in-protein-chemistry

the most frequently used are Agarose (more commonly for nucleic acids due to large pore size) and Polyacrylamide gels, which are more
frequently used for proteins.
Polyacrylamide Gel Electrophoresis: Polyacrylamide gels (Figure 8) are formed by the polymerization of acrylamide
(CH2CHCONH2), in the presence of free radical generator such as ammonium persulfate or riboflavin. The linear chains of polyacrylamide
are cross-linked with the help of N, N methylene bisacrylamide ([CH2CHCONH]2CH2). Gels of different pore sizes may be formed by
increasing or decreasing the percentage of monomers in the polymerization mixture. When the electric current is applied, proteins while
moving towards opposite electrode experience a sieve effect due to the pores of the gel. This allows smaller proteins to move faster than
the larger ones. For protein separation, virtually all methods use polyacrylamide that covers a size range of 5–250 kD. In PAGE, the gel
is mounted between two buffer chambers, current flows through the gel. The gels may be run both in vertical and horizontal modes. The
former is more common for polyacrylamide gels (Figure 9A).
Figure 8: Structure of polyacrylamide.
Two types of buffer systems can be used:
» Continuous buffer systems; which use the same buffer (at constant pH) in the gel, sample, and the reservoirs. Continuous systems
are not suitable for protein separations, and are used with nucleic acids.
» Discontinuous buffer systems; which use different buffer for reservoirs, the gel (sometimes two gels; a large pore stacking gel and a
resolving gel) and the sample. These are generally employed with proteins.
Following electrophoresis, the gel may be stained (most commonly with Coomassie Brilliant Blue R-250 or silver stain), allowing
visualization of the separated proteins, or processed further (e.g. Western blot). After staining, different proteins will appear as distinct
bands within the gel (Figure 9B).
The following sections take up some of the polyacrylamide gel electrophoresis techniques that are utilized for purification of proteins.
Native Polyacrylamide Gel Electrophoresis: In Native Polyacrylamide Gel Electrophoresis (Native PAGE), proteins are prepared
in a non-denaturing sample buffer, and electrophoresis is performed in the absence of any denaturing or reducing agents. Since the
native charge and mass of the proteins is preserved, the movement is primarily according to its charge to mass ratio. However, additional
factors (such as shape) that also influence the movement of proteins make the data from native PAGE quite difficult to interpret. Multisubunit
proteins may also be separated in their native form as the conditions are mild and non-denaturing. However, their movement
is even more unpredictable. Depending upon charge, protein may move towards either of the electrodes. Native PAGE cannot be used
for determination of molecular weight. Nonetheless, it does allow separation of proteins in their active state and can separate proteins
of the same molecular weight on the basis of charge differences. Overall, it is a low resolution technique for purification but still a useful
one due to discussed reasons.
PAGE may also be used as a preparative technique for the purification of proteins. For example, quantitative preparative native
continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is a method for separating native metalloproteins in complex biological
matrices.
Sodium Dodecyl Sulfate: Polyacrylamide Gel Electrophoresis: Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-
PAGE) was developed by Laemmli [15] in order to overcome the limitations of native PAGE. The detergent sodium dodecyl sulfate was
incorporated in the discontinuous polyacrylamide gel electrophoresis buffer system (hence the name SDS-PAGE). However, the loss of
native structure and biological activity in SDS-PAGE limits its use in condition where the isolation of protein in native state is desired.
In the presence of SDS (that disrupts all the non-covalent interactions in the protein) and a reducing agents (such as 2-mercapto
ethanol, which reduces and breaks disulfide linkages within a polypertide or between the subunits), they become fully denatured and
different subunits dissociate from each other. The non-covalent but uniform binding of SDS to proteins leads:
» An overall negative charge on the proteins by masking the intrinsic charge of protein.
» A uniform binding of SDS (1.4g of SDS per 1g protein, or approximately, one SDS molecule per two amino acid residues) generates
a similar charge-to-mass ratio for all proteins in a mixture.
OMICS Group eBooks
016