SIMULTANEOUS ANALYSIS OF CYCLIN AND ONCOGENE EXPRESSION USING MULTIPLE MONOCLONAL-ANTIBODY IMMUNOBLOTS

Cell dysfunction or dysregulation in cancer generally results from com plex gene interactions, numerous cellular events and environmental inf luences which modify gene expression or post-translational protein mod ifications. Genetic analysis in itself cannot always predict: or diagn ose multigenic diseases. The major technical difficulty is thus to def ect, identify and measure simultaneously the expression of several gen es and the post-translational modifications of their products. In orde r to progress io this direction, this paper describes a simple immunob lot method using several monoclonal antibodies to simultaneously analy ze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human live r biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophore sis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) m embranes. The membranes were stained with amido black, scanned and tes ted separately with the nine monoclonal antibodies p53, c-myc, PCNA, M EK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of inter est were then combined to form a mixture, and simultaneously used as t he primary antibobies. This antibody mixture simultaneously detected t he nine proteins of interest on both samples and it demonstrated the e xtensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.