High-resolution two-dimensional electrophoretic analysis of acidic, basic and surface proteins of mouse neutrophils.
The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 20 proteins detected) and with fluorography or autoradiography after [35S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of 125I associated with actin) yielded more than 20 major 125I-labelled proteins on high-resolution electrophoretic maps. The major 125I-labeled protein (Mr approximately 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [35S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.
D000199:Actins / Q000302:isolation & purification; D000818:Animals; D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000032:analysis*; D011506:Proteins / Q000302:isolation & purification*
D000199:Actins; D008565:Membrane Proteins; D011506:Proteins