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

Methods for Experimental Investigation of Protein Structure
Methods for Determining Protein Molecular Weight
Determination of Molecular Weight by Gel Filtration

The METHOD FOR DETERMINING the Molecular Weight of Proteins using Gel filtration is based on the following empirical fact. It has been established that the elution volumes of different proteins with identical molecular weights on the same Column under identical conditions are practically identical. This makes it possible to determine the molecular weight of an unknown protein if the gel column has been pre-graduated (calibrated). Calibration involves determining the elution volumes of several pure proteins with known molecular weights. The obtained data are plotted as the logarithm of MW versus the elution volume (Ve). Such relationships within a range of one to one-and-a-half orders of magnitude in molecular weight are typically linear. As an example, the figure above shows such a plot for a series of proteins.

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Instead of the elution volume, the partition coefficient (Kp) is sometimes used, which is calculated by the formula:

Kp = (Ve - Vвнеш) / Vвнутр

and then a calibration plot is constructed in the coordinates of the linear equation:

Kp = B - Alg MM

Deviations from linearity are observed for very large molecules, low-molecular-weight substances, and certain proteins, such as Collagen and fibrinogen.

For Globular proteins and many CARBOHYDRATES, the described method for determining molecular weight yields reliable results (= 10%).

When fractionating proteins in a thin layer of G-200 gel (on a plate), a linear relationship is observed between the relative migration distance of the protein spot and the logarithm of its molecular weight. The relative migration distance is calculated relative to the migration distance of a reference standard protein, such as Hemoglobin.

To obtain reliable results, gel filtration is recommended to be carried out in the presence of Denaturing Agents, such as in a concentrated solution of guanidine hydrochloride.



Last update: 06/08/2026

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