Parameters of the labeling of mitogen-activated murine lymphocytes by [35S]methionine for two-dimensional gel electrophoresis. I. Effect of culture conditions.
Labeling with [35S]methionine at a high specific activity is essential to the facile preparation of 2-dimensional gel electrophoretograms with the analytical 2-dimensional charge-size separation procedure (Anderson's ISODALT system). Mitogen-activated T and B lymphocytes subjected to low methionine concentrations would not proceed through cell cycle. In the case of activated B lymphocytes, the use of fetal bovine serum (FBS), dialyzed to lower endogenous methionine concentrations, prevented B cell growth even in the presence of otherwise satisfactory levels of methionine. High concentrations of [35S]methionine (greater than 300 mCi/1) induced B cell death, apparently by radiation damage. Despite these problems, good radioautograms and radiofluorograms of 2D electrophoretograms could be prepared by labeling activated B or T cells in bulk (10(6) cells/ml) with high specific activity [35S]methionine. The polypeptides labeled may be a biased sample since lymphoid cells do not proceed through cell cycle under these conditions. Small numbers (10(3] of activated T cells also yielded satisfactory samples but labeling of small numbers of activated B cells was not possible.
D000818:Animals; D001345:Autoradiography; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007526:Isoelectric Point; D008213:Lymphocyte Activation; D008715:Methionine / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*
D003470:Culture Media; D011506:Proteins; D008715:Methionine