Elimination of non-specific binding in western blots from non-reducing gels.

The reaction of some antibodies with Western blots of protein shows strong non-specific binding especially at a region that corresponds to about 70-90 kDa. This binding is independent of protein concentration. Further analysis indicated that the factor responsible for the non-specific binding is 2-mercaptoethanol in the gel sample buffer. Gel electrophoresis of total tissue homogenates in the absence of this reducing agent resulted in dramatic elimination of the non-specific background binding without affecting the mobility of the two proteins we studied.