You should load lesser amount of protein

I agree with Anirban and Ajay that lowering the protein conc. will definetly reduce the smearing.

Another things you can try is adding a reversible proteinase inhibitor to the sample buffer/running buffer, packing the gel rig in ice, running the gel faster or increasing the protein conc. in the gel preparation (if you are casting your own gels).

Thanks for the suggestions.....i will be grateful if you can suggest me some good and established protocols for zymogram of protease enzyme.

Well, here is an oldie. It has a good protocol if you are casting your own gel.

http://cnbr.mbi.ufl.edu/Faculty/wang/Raser25CaseinZymography-ABB1995.pdf

They used casein, but you can substitute that for any protein you want.

Yeahh..the protocol is good.

Could you provide a good reversable protease inhibitor and a protocol to use it? That's a question I am facing: lysing cells expressing a recombinant protease, keeping the enzyme active while avoiding degradation by native proteases.

Hi Luis. There is no single reversible inhibitor that will work against all proteinases.

Reversible inhibition you can do includes: buffers at pH ~7.5 against most aspartyl proteinsaes, EDTA (1-10 mM) for most metalloproteinases and substrate analogs such as benzamidine, lysine, tryptophan, etc. (1-10 mM) for most trypsin- and chymotrypsin-like serine- and sulfhydryl proteinases. Also, keep samples on ice for short-term storage and at -80oC for long-term storage.