I am working on recombinant subtilisin-like serine protease. As I have to analyze whether my recombinant protein of interest is active or not, I am using azocoll as substrate. I have used proteinase k as a standard (where I could see digestion of the substrate), but in the case of my protein, there is no increase in the colour to purple. I have also tried with bacterial crude lysate where I again found the same. According to some literature, the buffer I used is 20mM tris. Hcl and 2mM CaCl2. Incubation at 37C in shaker waterbath. Please help me find where the problem is.
Hi Sharmila,
azocoll is azo-impregrated insoluble collagen. We are testing this substrates only with collagenases/gelatinases. There is another, more universal substrate you should use - casein-resorufin (you can buy it from Roche, Sigma or Calbiochem). Collagen can be difficult to cleave by some proteases, as there could be a substantial amount of the triple-helical structure preserved. And this is onla a substrate for real collagenases!
Good luck, Magdalena
hey magdalena,
thank you very much for your response ... I will try the other substrate ...
please help me out.. I am also using azocoll (purple colour) for collagenase detection.. control is giving light purple colour whereas few test are giving dark purple colour while few test are giving light red colour. so, i am confused which are the positive one???
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