Biochemical and immunological identification of human neutrophil elastase on nitrocellulose membranes.
Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine alpha-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 microgram HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils.
Takeuchi
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Roswell Park Memorial Institute, Molecular and Cellular Biology Department, Buffalo, New York 14263.
D015153:Blotting, Western; D002244:Carbon; D003101:Collodion; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019272:Leukocyte Elastase; D008567:Membranes, Artificial; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000032:analysis* / Q000276:immunology; D011506:Proteins / Q000032:analysis* / Q000235:genetics; D012394:Rosaniline Dyes; D013194:Staining and Labeling
D004396:Coloring Agents; D008567:Membranes, Artificial; D011506:Proteins; D012394:Rosaniline Dyes; C028433:chinese ink; D002244:Carbon; D003101:Collodion; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; C004692:coomassie Brilliant Blue