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 (> 300 mC_i/l) 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 (10⁶ 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 (10³) of activated T cells also yielded satisfactory samples but labeling of small numbers of activated B cells was not possible.