Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel.
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 degrees 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.
Choi
Nack-Shick
NS
Enzyme Based Fusion Technology Research Team, Jeonbuk Branch Institute, Jeonbuk, Korea.
Choi
Jong Hyun
JH
Kim
Bo-Hye
BH
Han
Yun-Jon
YJ
Kim
Joong Su
JS
Lee
Seung-Goo
SG
Song
Jae Jun
JJ
D001407:Bacillus / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism; D008049:Lipase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D009391:Nephelometry and Turbidimetry; D010447:Peptide Hydrolases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012394:Rosaniline Dyes; D013210:Staphylococcus / Q000201:enzymology; D014280:Triglycerides / Q000737:chemistry* / Q000378:metabolism
D012394:Rosaniline Dyes; D014280:Triglycerides; C048139:Coomassie blue; D005337:Fibrin; D008049:Lipase; D010447:Peptide Hydrolases; C005830:tributyrin