Abrogation of c-MYC protein degradation in human lymphocyte lysates by prior precipitation with perchloric acid.
Conventional lysis buffers, though containing cocktails of protease inhibitors, did not prevent the degradation of c-MYC recombinant protein added immediately prior to lysis to cell pellets from human mixed lymphocyte cultures. Treatment of the cells with 4.2% perchloric acid, however, prevented protein degradation and facilitated the detection of c-MYC protein by Western blotting even in unstimulated lymphocytes, where previously it had been reported to be undetectable or barely detectable using this technique. PHA stimulation of lymphocytes induced an approximately six fold increase in measured c-MYC protein within 5 h if cell extracts were prepared using perchloric acid precipitation. However, using conventional lysis buffer the proto-oncogene protein was undetectable until 48-72 h after mitogen addition. Pretreatment with perchloric acid may be useful for Western blotting analysis of protein in other systems where it may be desirable to dispense with the use of toxic protease inhibitors or where these may be incompletely effective.
Spiller
D G
DG
Department of Biochemistry, University of Liverpool, UK.
Tidd
D M
DM
D015153:Blotting, Western / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D003720:Densitometry; D005434:Flow Cytometry; D006801:Humans; D010472:Perchlorates; D010835:Phytohemagglutinins; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000302:isolation & purification*; D013997:Time Factors
C000715470:MAS1 protein, human; D010472:Perchlorates; D010835:Phytohemagglutinins; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D004247:DNA