It is true that the diffusion of soluble chromophores or fluorophores wil limit the resolution of zymograms. However, diffusion is a slow process, and if the reaction is sufficiently fast, you will still be able to detect the location of the enzyme.  MUG would be a good substrate in this respect, since  detection of methylumbelliferone is highly sensitive, thus limiting the incubation time required for detection.

Hello Deepti

Well, monitoring the secretion pattern of cellobiase using a zymogram may be a difficult hurdle due to the solubilization problem as you have mentioned. However, I hope there is a way out to this challenge. I am still brainstorming on it because it could be a wonderful breakthrough due to its conveniency

Best of luck

MUG is the best substrate to do the zymogram of BGL, just ensure you take optimum MUG concentration and for the specified time. Once reaction is over immediately transfer the gel to a good gel-doc system ready to take pic. Timing is crucial, otherwise you may get faint bands.

Good luck!!!

I would worry less about the diffusion of the enzymes (and proteins in general) and more about the diffusion of the product. The way we deal with this is by instead of immersing the gel in substrate solution we place a sheet of  Whatman 3 filter paper on the gel and soak it with substrate solution.

Yoram

Thanks Pierre and Jitendra for your valuable suggestions.... We are working on the optimization of the same.