Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.