I have done zymography of amylase, in which i followed the SDS solution protocol, but now when i did it for protease, i again followed the same protocol,
In the end i use 10 mM tris buffer for washing SDS, can anyone share protease zymography protocol.
Thanks
how its possible ! because their SDS is denaturing agent. so, u can select wright method for zymogram of protease.
is it source of plant, microbial, animal or insect. whatever ur enzyme source u can try native gel
thanks
u can try by using native PAGE and replace amylase substrate with casiene
Here is a link for zymogram protocol.
http://www.millipore.com/userguides/tech1/mcproto009
There are also precast zymogram gels (invitrogen), casein and gelatin (if you don't know the kind of protease I recomend do both gels). Do not use betamercapto or DTT before inject the sample on the gel. After you finish the running of the gel you need to use the renaturing buffer (Triton) to eliminate all the SDS present and then you can incubate your gel in the tris buffer at 37C to activate the protease (overnight or at least 4h, this depends of the type of protease and concentration). Then you do the staining with coomassie
Thanks for all of you, i have done the protease zymography, with native SDS PAGE.
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