Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Methods for Experimental Study of Protein Structure
Methods for Determining Protein Molecular Weight
Determination of Protein Molecular Weight by SDS-PAGE

Sodium dodecyl sulfate (SDS) is a strong denaturing agent. The Use of SDS makes it possible to impart a similar Structure to all protein molecules while preserving differences in their molecular weight.

In a polyacrylamide gel at neutral pH in 1% SDS and 0.1 M mercaptoethanol, most multi-subunit Proteins bind SDS and dissociate. The existing Disulfide Bonds are thereby cleaved by mercaptoethanol. The Introduction/11.html">Secondary structure of the protein is disrupted, As a result of which the protein-SDS complexes acquire a random coil structure; that is, after Treatment with SDS, the molecules of all proteins acquire approximately the same structure and have a uniform charge-to-mass ratio. This is due to the fact that The amount of SDS bound to the protein is relatively constant, at about 1.4 g of SDS per 1 g of protein. Consequently, the charge of the SDS-coated protein molecule is determined practically not by The amino acid residues of the peptide chain, but by the surface-bound SDS molecules, which readily dissociate In aqueous solutions and carry a negative charge.

Hydrodynamic studies have shown that protein-SDS complexes are prolate ellipsoids or rods of constant cross-section, with a diameter of = 18Å.

It has been established that during gel Electrophoresis of protein-SDS complexes, There is a linear relationship between their electrophoretic mobility (μ) and the logarithm of the molecular weight:

Class="center">μ = b - alg MM

The existence of this relationship makes it possible to determine the Molecular Weight of unknown proteins using calibration curves within the appropriate mass range. The error in determining molecular weights by this method is about 10%.