Electrotransfer of basic proteins from nondenaturing polyacrylamide acid gels to nitrocellulose: detection of enzymatic and inhibitory activities and retention of protein antigenicity.
We have developed a method for electrotransfer of strongly basic proteins (lysozyme, pI 11; mucus proteinase inhibitor, pI greater than 10; bovine pancreas trypsin inhibitor; pI 10.5; human leukocyte elastase, pI greater than 9) from nondenaturing acid gels (pH 4.5) to nitrocellulose sheets. Buffers were those used in a discontinuous system for transfer from sodium dodecyl sulfate (SDS)-containing polyacrylamide gels with one modification in the cathode buffer which contained 0.1% SDS. This method was compared to electrotransfer performed in 0.7% acetic acid. The basic proteins studied, which were positively charged in the gel, formed with SDS negative complexes which migrated toward the anode and were efficiently transferred to the nitrocellulose. Moreover, their biological properties were preserved: inhibitory activity, enzyme activity, and antigenicity. This method is advantageous because it is simple, is sensitive, and can be applied to various biological fluids to detect inhibitors, enzymes, and other proteins which have a basic character, after electrophoretic separation under their native forms.
Van-Seuningen
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I
Unité INSERM No. 16, Lille, France.
Davril
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M
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D056649:Cathepsin G; D002403:Cathepsins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000032:analysis / Q000276:immunology; D010196:Pancreatic Elastase / Q000032:analysis; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000032:analysis* / Q000276:immunology; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000032:analysis; D013183:Sputum / Q000201:enzymology; D013194:Staining and Labeling; D014361:Trypsin Inhibitors / Q000032:analysis
D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D009113:Muramidase; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase