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Mixed‐substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel
Author(s) -
Choi NackShick,
Choi Jong Hyun,
Kim BoHye,
Han YunJon,
Kim Joong Su,
Lee SeungGoo,
Song Jae Jun
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800727
Subject(s) - zymography , coomassie brilliant blue , bacillus licheniformis , chemistry , chromatography , glycerol , proteolytic enzymes , gel electrophoresis , substrate (aquarium) , biochemistry , fibrin , enzyme , polyacrylamide gel electrophoresis , two dimensional gel electrophoresis , electrophoresis , biology , staining , bacteria , proteomics , bacillus subtilis , ecology , gene , genetics , immunology
A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS‐containing or native‐conformation gel and a mixed‐substrate (glycerol tributyrate and fibrin) (MS) 1 gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X‐100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37°C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ‐1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ‐2 and B. licheniformis NJ‐5, isolated from soil, can be simultaneously detected.