Analysis of protein incorporation of radioactive isotopes in the chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis

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Abstract
The patterns of [3H]‐leucine and [32P]‐phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two‐dimensional polyacrylamide gel ultra‐microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of [3H]‐leucine‐ and [32P]‐phosphate‐labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.