Ligand western blotting for specific detection of active forms of proteases.
We developed a non-radioactive method of ligand western blotting for specific detection of active forms of serine proteases. The method consists of three steps: (i) separation of proteins by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel, followed by blotting of proteins to nitrocellulose membrane; (ii) binding of a specific ligand, such as soybean trypsin inhibitor labeled with biotin, to protease on the membrane; and (iii) detection of the protease-inhibitor complex by color reaction (or chemiluminescence) developed by streptavidin-conjugated peroxidase (or alkaline phosphatase). By using this method, plasmin and trypsin (serine proteases) were detected, but papain (thiol protease) or pepsin (acidic protease) was not. Plasmin was detectable up to less than 4 ng. Inactive precursors of serine protease, i.e. plasminogen and trypsinogen, did not exhibit visible bands until they were activated by treatment with streptokinase or trypsin, respectively. We applied this method to clinical samples, and succeeded in detecting plasminogen, after conversion to plasmin with streptokinase treatment, in as little as 5 microliters of serum or trypsin, as it was in 10 microliters of pancreatic juice.
Kido
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Division of Medical Technology, College of Medical Technology, Kyoto University, Japan.
Yook
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Ueda
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D001426:Bacterial Proteins; D001710:Biotin; D015153:Blotting, Western / Q000379:methods* / Q000706:statistics & numerical data; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D005341:Fibrinolysin / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D008832:Microchemistry; D010189:Pancreatic Juice / Q000201:enzymology; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000032:analysis* / Q000302:isolation & purification; D019809:Streptavidin; D014357:Trypsin / Q000032:analysis / Q000097:blood; D014361:Trypsin Inhibitors / Q000378:metabolism
D001426:Bacterial Proteins; D014361:Trypsin Inhibitors; D001710:Biotin; D003101:Collodion; D019809:Streptavidin; D012697:Serine Endopeptidases; D014357:Trypsin; D005341:Fibrinolysin