Question

Can Western blotting be used as a quantitative technique? How could we use Western analysis to determine the relative change in a protein's expression levels between two treatments? What would we need to be able to determine the absolute level of a protein (i.e. ng/mg of total protein)?

Answer 1

Western Blot is a technique that is used to separate on a polyacrylamide on the basis of their size. This technique can also be exploited to quantify the amount of proteins present in a sample. However, this cannot be done directly and an appropriate strategy needs to be used for the same.

1. If we want to look at the relative protein expression levels between two treatments, we can do so by performing Western blot for the protein in two treatment conditions. The image can then be analyzed using Softwares like ImageJ and the intensity of the bands can be quantified. The ratio of the intensities between the two treatment conditions will give a value of the relative change of proteins in two different conditions.

2. To determine the absolute concentration, a standard plot is required. Here, we have to load different known concentrations of the protein on a Western blot along with the unknown sample. The intensities of the band in each of the scenarios can then be recorded using the software. This can then be solved using a linear regression model and then this model can be used to predict the concentration of an unknown sample. Thus, the absolute concentration can be obtained by this method.