The protooncogene c-Myc plays a key role in growth control, differentiation
, and apoptosis. An abnormally high expression of c-myc has been found to b
e associated with many neoplasms. c-Myc gene expression is usually measured
at the mRNA level Few studies have been published on quantitative Myc prot
ein determination. A major drawback of ELISA (enzyme-linked immunosorbent a
ssay) methods is the uncertainty of the specificity of the antibody reactio
n. In contrast, antibody specificity can be easily controlled by Western/im
munoblotting. Here we describe a method to quantify c-Myc protein in primar
y human IMR90 lung fibroblasts based on Western blotting Using a high-resol
ution polyacrylamide gel, we were able to differentiate the cellular c-Myc
protein (64 kDa) from a c-Myc internal standard (65 kDa). We determined bot
h the total c-Myc protein content per cell and its distribution in the cyto
plasmic and nuclear fractions. About 4000 c-Myc protein molecules were dete
cted in the cytoplasmic fraction and 29,000 copies in the nuclear fraction
for proliferating human limp fibroblasts IMR90. The ratio of nuclear (activ
e) to cytoplasmic (inactive) c-Myc protein changed from 17:1 for proliferat
ing cells to 2.5:1 for confluent cells. (C) 1999 Academic Press.