Identification of a higher molecular weight protein that shows apparent cross-reactivity with anti-p21ras monoclonal antibodies on western blots.

We have characterized the immune cross-reactivity of several commercially available monoclonal antibodies prepared against the p21ras proteins and used in Western blotting experiments against human tissue homogenates. Under optimal conditions, only two bands were observed on Western blots. One of these comigrated with control p21ras protein. A second protein of apparent mobility corresponding to approximately 54 kDa was also observed with all four monoclonal antibodies tested. Protein sequencing by automated Edman degradation indicates that the 54 kDa species corresponds to human immunoglobulin heavy chain. Under suboptimal conditions, another high molecular weight species of apparent mobility 65 kDa was also observed to cross-react with some of the monoclonal antibodies tested. This 65 kDa species was identified by protein sequencing as human serum albumin. Coomassie blue staining of SDS-polyacrylamide gels indicates that serum albumin is a major contaminant of many surgically obtained human tissue samples, while p21ras and immunoglobulin heavy chain are present at much lower concentrations. These results may be of significance when using monoclonal antibodies to determine p21ras levels of whole tissue homogenates by dot-blot, slot-blot or microplate assays.