Comparison of 14C-amino acid mixture and [35S]methionine labeling of cellular proteins from mouse fibroblast C3H10T1/2 cells by two-dimensional gel electrophoresis.
Total cellular proteins from mouse C3H10T1/2 fibroblasts were compared by two-dimensional (2-D) gel electrophoresis after radiolabeling with [35S]methionine (35S-Met) or 14C-amino acids (14C-AA). 35S-Met labeling of protein was three to four times greater than 14C-AA incorporation over a 24 h period. Automated comparative analysis of replicate fluorographs after 6, 12, and 24 h of labeling showed considerable homology between radiolabeling methods. More than 88% percent of 35S-Met and 14C-AA-labeled proteins were common at each time point. However, the total number of 35S-Met-labeled proteins dropped from 6 to 24 h while the number of 14C-AA-labeled proteins increased. Additionally, twenty-one proteins were uniquely labeled by 14C-AA that were not detectable by 35S-Met over the labeling period. Densitometric analysis showed that several 35S-Met and 14C-AA-labeled proteins exhibited time-related differences in radiolabel incorporation while most proteins remained relatively constant. Protein patterns of silver-stained gels from 6 to 24 h were highly registered and showed few qualitative differences. Proteins detected in radiolabeled gels were generally, but not always, found in silver-stained gels. Thus, 35S-Met appears better suited for short-term radiolabeling of cellular protein while more comprehensive labeling of protein occurs with 14C-AA during prolonged incubation of cell cultures under present experimental conditions.
He
C Y
CY
Division of Toxicology Research and Testing, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Merrick
B A
BA
Mansfield
B K
BK
Hite
M C
MC
Daluge
D R
DR
Selkirk
J K
JK
D000596:Amino Acids; D000818:Animals; D002250:Carbon Radioisotopes; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D008715:Methionine; D051379:Mice; D010779:Photofluorography; D011506:Proteins / Q000032:analysis*; D016622:Silver Staining; D013462:Sulfur Radioisotopes
D000596:Amino Acids; D002250:Carbon Radioisotopes; D011506:Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine