I am working on Bacillus sp. which shows the cellulolytic activity. After partial purification of the cellulase enzyme i am incorporating 0.2 % CMC in the PAGE (Native) gel and running it at 4 degree celcius. But after staining it with congo red, it shows a trail of activity not a specific spot around the band. Please help me whether there are any other methods for CMCase zymography?
The reason you get a trail instead of a band is that your enzyme is digesting CMC all along during migration. If you want to avoid this, you should transfer the enzyme after migration to a CMC-containing agar replica and then stain the latter with Congo red. See Beguin P. 1983 Anal Biochem 131,333-336 (unfortunately, I no longer have reprints nor access to this paper to forward to you).
Dear Mudasir,
You can use these following papers for your CMCase zymography assay
Mr. Beguin, Thank you sir, i have already tried the same thing by using 0.5 and 1% CMC in agar slabs /plates, but unfortunately the trail kind of activity still appears. Even I optimized the CMC concentrations as well as incubation period for the zymogram activity.
Thanks Mahesh Bhai, already gone through these protocols. Actually the other problem is Mine Cellulase is not completely purified, it is only partially purified....
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