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 ...
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