Determination of copy number of c-Myc protein per cell by quantitative Western blotting.
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 be associated with many neoplasms. c-Myc gene expression is usually measured at the mRNA level. Few studies have been published on quantitative Myc protein determination. A major drawback of ELISA (enzyme-linked immunosorbent assay) methods is the uncertainty of the specificity of the antibody reaction. In contrast, antibody specificity can be easily controlled by Western/immunoblotting. Here we describe a method to quantify c-Myc protein in primary human IMR90 lung fibroblasts based on Western blotting. Using a high-resolution polyacrylamide gel, we were able to differentiate the cellular c-Myc protein (64 kDa) from a c-Myc internal standard (65 kDa). We determined both the total c-Myc protein content per cell and its distribution in the cytoplasmic and nuclear fractions. About 4000 c-Myc protein molecules were detected in the cytoplasmic fraction and 29,000 copies in the nuclear fraction for proliferating human lung fibroblasts IMR90. The ratio of nuclear (active) to cytoplasmic (inactive) c-Myc protein changed from 17:1 for proliferating cells to 2.5:1 for confluent cells.
Rudolph
C
C
Fakultät für Biologie, Universität Konstanz, Konstanz, D-78457, Germany. Claudia.Rudolph@uni-konstanz.de
Adam
G
G
Simm
A
A
D015153:Blotting, Western / Q000379:methods*; D002478:Cells, Cultured; D006801:Humans; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis* / Q000276:immunology; D012015:Reference Standards; D012044:Regression Analysis
D016271:Proto-Oncogene Proteins c-myc