Determination of Molecular Weight of a Protein by Gel Filtration

Introduction Protein purification is an indispensable technique for providing biochemists with the ability to obtain samples containing specific target proteins. To purify the protein, the biochemist usually obtains a particular extract, precipitates the protein using ammonium sulfate, preforms gel filtration and finally performs ion chromatography and affinity chromatography . Understanding the usage and method of different chromatographic techniques is important for successful purification of unknown samples and estimation of molecular weight using elution volumes of molecular weight standards.

Size exclusion chromatography, also known as gel filtration or gel permeation chromatography, uses porous particles to separate molecules of different sizes. It is commonly used to separate biomolecules and to determine the molecular weight and molecular weight distribution of the polymer. Molecules smaller than the pore size can enter the particle and thus have a longer path and longer transit time than larger molecules that can not enter the particle. Molecules larger than the pore size can not enter the pores and elute as the first peak in the chromatogram. This situation is called full exclusion. The molecules that can enter the pores will have an average residence time within the particles depending on the size and shape of the molecules. Therefore, the total transit time through the column varies depending on the molecule. Selective transmission region

All cellular proteins can be separated by SDS gel electrophoresis (eg 42 kDa protein to 41 kDa protein) to isolate proteins with relatively large molecular weight differences but not similar molecular weight proteins. In order to isolate similar quality proteins, other physical properties must be utilized. Most commonly this is a charge, which is determined by the number of acidic and basic residues in the protein. Two unrelated proteins of similar quality are unlikely to have the same net charge due to their sequence and hence the number of acid and basic residues