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 Molecular Weight by Sedimentation - Sedimentation Equilibrium Method

In the Sedimentation Equilibrium method, the rotor speed of the centrifuge is adjusted so that the opposing forces of sedimentation and diffusion balance each other. Once steady equilibrium is reached, no pure solvent zone remains near the meniscus. A protein concentration gradient is established along the centrifuge tube, with the concentration in the bottom layer potentially reaching roughly twice that in the top layer. By measuring the protein concentration at various distances from the axis of rotation, its molecular weight can be calculated using the following equation:

Class="center">М = 2RTln(c2/c1)/ω2(1 - vp)(r22 - r12)

where c2 and c1 are the protein concentrations in the tube at distances r2 and r1 from the axis of rotation, respectively.

In this approach, calculating the molecular weight does not require knowing the diffusion coefficient. Furthermore, the sedimentation equilibrium method requires no corrections for particle shape, making it the most accurate of all sedimentation techniques. Its primary drawback is the substantial time investment required; establishing equilibrium sometimes necessitates continuous centrifugation for several days. This process can be accelerated by using Cells with a very short liquid Column height. Additionally, the sedimentation equilibrium method is highly sensitive to protein purity and homogeneity.



Last update: 06/08/2026

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