Two-dimensional electrophoresis and densitometric analysis of solubilized bovine sperm plasma membrane proteins detected by silver staining and radioiodination.
Bovine sperm plasma membranes were extracted with deoxycholate and subjected to two-dimensional gel electrophoresis. Proteins were visualized either by silver staining or autoradiography using 125I. When 1.5-2.0 micrograms of protein extract was applied to the first-dimension get, 250 spots could be detected by autoradiography. Thirty micrograms of protein was required to obtain spot visualization using silver strain, revealing more than 500 spots on the gel. These data establish the limit under which two-dimensional electrophoresis can be used in conjunction with flow cytometric sorting of sperm for the analysis of possible sex-specific membrane differences. The necessity of orienting sperm as they past the laser in the cell sorting system reduces throughput; thus, the number of sperm sorted within a given time is limited. It is suggested that autoradiography will allow the flow cytometer to be used in a time-efficient manner. Longer sorting times would be required to obtain sufficient sample to analyze total protein composition.
Fenner
G P
GP
U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705.
Johnson
L A
LA
Hruschka
W R
WR
Bolt
D J
DJ
D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D003720:Densitometry; D004229:Dithiothreitol; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D007457:Iodine Radioisotopes; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008970:Molecular Weight; D012834:Silver; D012995:Solubility; D013094:Spermatozoa / Q000737:chemistry* / Q000648:ultrastructure; D013194:Staining and Labeling
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D012834:Silver; D004229:Dithiothreitol