A Zymographic Method for Screening Microbial Extracellular Collagenase

Abstract

The invention discloses a zymogram method for screening microbial extracellular collagenase. The collagenase is subjected to zymogram analysis by utilizing a specific degradation effect of the collagenase on collagens. Gel adopted in the electrophoresis process is prepared by adding type I collagen with the mass volume ratio concentration of 0.1 percent into 10 percent of SDS-PAGE gel. The gel treatment process comprises the following steps: oscillating and diluting the gel firstly by using eluent after the electrophoresis is ended, then oscillating and bleaching by using a bleaching solution, and finally standing and incubating the gel at the temperature of 37 DEG C by using an incubating solution for 12 hours; staining the gel for an hour by using a Coomassie brilliant blue R-250 staining solution after the incubation is ended, and performing room-temperature oscillating and discoloring through a destaining solution until the transparent strip is clear. According to the method, the type I collagen is prepared into the SDS-PAGE gel, the final concentration of the type I collagen in the gel is 0.1 percent, the collagenase negative staining strip and the standard protein standard strip can be simultaneously developed on the gel under the substrate concentration, and the technical gap in the collagenase zymogram detection field is filled.

Description

technical field [0001] The invention belongs to the technical field of biological protease detection, and in particular relates to a zymography method for screening microbial extracellular collagenase. Background technique [0002] Zymography analysis technology is developed on the basis of sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis. Zymography analysis technology refers to the use of gels containing substrate proteins for electrophoresis or soaking gels without substrate proteins in the substrate solution after electrophoresis, and then through incubation, staining, and decolorization methods to display Changes in the staining of the substrate protein, and then qualitative analysis of the enzyme activity. [0003] Although the method of zymography analysis is often used to detect the activity of proteases, the existing zymography technology still has the following technical defects: [0004] (1) The current zymography technology is to detect the ...

AI Technical Summary

Problems solved by technology

However, the high concentration of substrate protein in the gel will affect the mobility of the protease in the gel, which may lead to a large deviation in the calculation of the molecular weight of the protease

[0006] (3) If the method of soaking the gel in the substrate solution is adopted, the resolution of the transparent band of the protease is likely to be low, and the band is not concentrated enough and clear

[0007] (4) NaN is commonly used in the gel incubation process in the current zymography analysis 3 As a preservative to avoid the influence of enzyme-producing microorganisms on the results, the effect is good but NaN 3 highly toxic and dangerous

Current zymography techniques cannot be applied to collagenase

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