I'm trying to make zymograms to evaluate endoglucanase activity, but had no luck. I have tried several protocols using a 0.2 IU per well in gels 1 mm SDS-PAGE and Native containing CMC, incubating for 30 min and 60 min at 50 ° C in citrate phosphate buffer 0.1 M pH 5 to carry out the reaction and using congo red for tinsión, and 1 M NaCl for washing for 1 hour.
Can anyone suggest to me any alternative protocol? 0.2 IU CMCase per well is sufficient to visualize degradation halo?
I suggest that you first conduct some preliminary experiments to know the conditions that work best for your endoglucanase e.g. best pH, temperature and enzyme concentration. It could be that the conditions you are using are not favourable for the enzyme activity. After getting the optimal conditions in your preliminary tests, then use those conditions in the zymogram analysis. Wishing you luck.
Thank you for your suggestion.
I performed tests pH and temperature optima eh also tried with various concentrations of enzymes. being the most high CMCase 0.2 IU per well.
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