Determination of native molecular weight of a protein using gel filtration chromatography
The molecular weight and size of a protein is related to the shape of the molecule and the relationship between molecular weight (M) and radius of gyration (Rg)is as follows-
here “a” is a constant and it depends on shape of the molecule, a=1 for Rod, a=0.5 for coils and a=0.33 for spherical molecules.
The set of known molecular weight standard protein can be run on a gel filitration column and elution volume can be calculated from the chromatogram (Figure 32.4). A separate run with the analyte will give elution volume for unknown sample. Using following formula, Kd value for all standard protein and the test analyte can be calculated.
A plot of Kd versus log mol wt is given in Figure 32.4, B and it will allow us to calculate the molecular weight of the unknown analyte.
Figure 32.3: Determination of molecular weight by gel filtration chromatography. (A) Gel filtration chromatogram with the standard proteins (1-6), (B) Relationship between distribution constant (Kd) and Log Molecular weight. |