lSodium dodecyl sulphate-polyacrylamide gel electrophoresis or SDS-PAGE is commonly used in protein aboratories for separating proteins based on their molecular weights. This is accomplished by taking advantage of their differential rates of migration through a sieving matrix (the acrylamide or agarose gel) in response to an applied electrical field.
The movement of charged particles through an electric field is determined by three factors - the net charge, the molecular radius and the magnitude of the applied field. However, in the case of native proteins, the net charge is determined by the amino acid composition (the sum of the positive and negative amino acids in the protein), and not by their molecular weight. In addition, the molecular radius is largely determined by the protein’s complex tertiary structure.
Thus, if we are to separate proteins in an electrical field without altering their native state, different proteins with the same molecular weight would then migrate at different speeds. Needless to say, the data resulting from native PAGE can be extremely difficult to interpret. Since the protein’s native charge is preserved, the molecules can migrate toward either electrodes, producing unpredictable separation patterns that are highly unsuitable for molecular weight determination.
To improve the quality of your results, you need to destroy the tertiary structure and impart a uniform net charge to your protein sample. And this is precisely the reason why the SDS-PAGE technique was developed.
Understanding the Role of SDS in SDS-PAGE
SDS is basically an alkyl sulfate, anionic detergent that is frequently used in protein electrophoresis and protein solubilization methods. Since SDS carries a highly negative charge and has a hydrophobic tail that interacts strongly with the protein or polypeptide chains, it can imparta relatively equal negative charge to all the protein molecules in the sample. Keep in mind that each SDS molecule binds with two amino acids. As such,it is so easy to see how it can overwhelm any charge the protein may have.
Aside from imparting a uniformly negative charge, SDS can also change the complex tertiary conformation of the protein molecules and transform them into long, rod-like molecules by disrupting the non-covalent protein-protein interactions that contribute to protein folding.
Now that your protein of interest has a net negative charge and a linear conformation, the rate at which they will migrate in a gel will primarily dependon its size.
The Low-Down on SDS
While the use of detergents such as SDS proves to be quite beneficial for initial cell lysis or membrane protein extractions, you may need to remove some or all of the detergent for subsequent applications or experiments using the proteins extracted using this technique.
There are cases wherein detergent solubilization modifies and disrupts the native lipid interactions, thereby rendering the membrane proteins inactive. However, in some of these cases, membrane protein function can be fully restored after the detergent is replaced with phospholipids or other membrane-like lipid mixtures.
Additionally, the reduction of detergent concentration is also necessary to increase your proteins’ compatibility with protein assays or gel electrophoresis.
So, how do you facilitate the removal of detergents from your sample solution? You can do this by dialyzing your sample, or by using ion exchange chromatography and/or sucrose density gradient separation. However, please bear in mind that while dialysis can be very effective in removing detergents that have very high CMCs and/or small aggregation numbers (such as the N-octyl glucosides), it cannot be used for detergents with low CMCs and large aggregation numbers. This is because most of the detergent molecules will be in micelles that are too large to diffuse through the pores of the dialysis membrane. In such cases, only excess monomer can be dialyzed.
Specialized detergent removal systems may also be used to serve this purpose without causing significant loss of proteins, dilution of the protein solution, or change to the buffer composition of the protein solution.
Image Source : Steve Jurvetson
