Determination of copy number of c-Myc protein per cell by quantitative Western blotting

Citation
C. Rudolph et al., Determination of copy number of c-Myc protein per cell by quantitative Western blotting, ANALYT BIOC, 269(1), 1999, pp. 66-71
Citations number
48
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
00032697 → ACNP
Volume
269
Issue
1
Year of publication
1999
Pages
66 - 71
Database
ISI
SICI code
0003-2697(19990410)269:1<66:DOCNOC>2.0.ZU;2-7
Abstract
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.