Gamma globulin-derived standards for the determination of molecular weights, transfer, and immunodetection efficiencies in protein blotting procedures

Molecular weight markers which are detectable using labeled antispecies antibodies or labeled Protein A have been prepared for use as standards on protein blots. The standards were prepared by the controlled reduction followed by subsequent alkylation of gamma globulin. Separate sets of standards were prepared using gamma globulins derived from human, mouse, rabbit, and sheep species. Standards were also prepared using monoclonal-derived gamma globulins from human myeloma fluid and mouse ascites fluid. Standards produced from monoclonal-derived gamma globulins produced very sharp bands on sodium dodecyl sulfate-polyacrylamide gels and proved to be excellent standards for this technique alone. However, the markers were uniquely suitable for use as standards in protein blotting procedures because their detection was achieved by the procedure used to detect the transferred antigen(s). The detection of immunoglobulin G (IgG)-derived standards on protein blots from all the species listed above was demonstrated using appropriate horseradish peroxidase (HRP)-conjugated antispecies antibodies. The use of other detection systems (biotin-labeled antibody and subsequent detection with HRP-steptavidin, HRP-Protein A) was also validated with human IgG-derived standards. Furthermore, the standards were shown to be suitable for use on both nitrocellulose and cationized nylon-based supports and could be used when adjacent samples were run under reducing conditions. Hence the gamma globulin-derived standards serve as both a control to check the adequacy of transfer and immunodetection systems and as markers which enable the molecular weights of detected antigens to be calculated.