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

Abstract

~ ~The patterns of [.3HI-leucine and [32Pl-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 GO + GI and (& + 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 ['HI-leucine-and ['2P]-phosphate-labeled protein regions from the autoradiographs occurring primarily inThe refinement of two-dimensional polyacrylamide gel electrophoresis (PAGE) has contributed significantly to the knowledge and the elaboration of gene products during cellular development and differentiation. In disciplines such as biochemistry, cell biology, teratology, etc., a need has arisen for methods to analyze single cells (eggs), small numbers of simple populations (e.g., cell cycle compartments, blastulae), and larger but relatively small numbers of complex populations (e.g., organs, embryos). The adaptation of a two-dimensional PAGE system for the analysis of minute amounts of protein from small samples has been developed (13), and the sensitivity was shown to be far greater than conventional electrophoresis techniques. For example, several hundred protein regions have been observed from a single Drosophila egg with resolution as low as 3 pg of protein (13).The objectives of the present study were, first, to assess the potential of micro-sampling for the collection of relatively small numbers of nuclei (200,000) from winthe 6 2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the Gz + 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 Gz + M and early mitosis were composed, in part, of nucleolar proteins.

The homology of a novel polypeptide with stress protein characteristics in embryonic mice brain and in the hypothalamus of caloric restricted rats as determined by ultramicro western blotting

“…Nuclei were sorted [23,24] on the basis of DNA quantity (PI fluorescence) using the FACStar Plus cell sorter (Becton-Dickinson, Mountain View, CA). The sorted samples of nuclei were used for protein analysis [21,25).…”