To investigate the cross-migration of proteins during nuclear isolation heated and control cells were mixed prior to nuclear isolation. These nuclei were stained with fluorescein isothiocyanate (FITC, a protein-specific stain) and with propidium iodide (PI, a DNA-specific stain). Flow cytometric (FCM) analysis showed two populations distinguishable on the basis of protein content. The protein content of the nuclei in the upper population was identical to that for nuclei isolated from heated cells while that for the lower population had a protein content identical to the protein content of nuclei from control cells. This result shows that the heat-induced increase in nuclear protein content occurred throughout the entire population of nuclei (i.e., in G1, S, and G2 nuclei) and that the measured protein content of nuclei was not affected by the presence of the other population during isolation. The capability of the FCM to sort subpopulations from different regions of a histogram was used to separate the subpopulations after analysis. When control cells were prelabeled with [3H]leucine and mixed with unlabeled heated cells, 11% of the radioactivity was found to be associated with the nuclei from heated cells. Autoradiographs showed grains over approximately 99% of the nuclei from heated cells. When [3H]TdR was used as a label in a similar experiment, only 0-3% of the label was observed to become associated with the population of nuclei from heated cells and autoradiography showed that 97% of these nuclei were not labeled. Comparable results were obtained when the labeled cells were heated and the control cells were left unlabeled. These results show that a small amount of protein (approximately 10% of the nuclear protein) will cross-migrate during nuclear isolation without affecting the net amount of protein in either population.
Cell Fractionation Cell Nucleus