On the masking of signals on immunoblots by cellular proteins

When determining the concentration of a particular protein in a cell extract, or when comparing the amount of a protein in different samples, it is a common practice to use specific antibodies in immunoblotting to compare the samples side by side with known amounts of purified protein. Here we show that with many antibodies, in particular monoclonal antibodies, the sensitivity of detecting the cognate antigen on immunoblots can be significantly reduced when the antigen is in a mixture with other cellular proteins. The signals on the immunoblots are masked by other endogenous proteins in the cell lysate, making the amount of the protein on the immunoblot appear to be less than the actual amount, thus invalidating direct comparison with purified protein.