When I run SDS PAGE gel and perform zymogram with same gel.I observed that the bands which are more intense (more protein) after CBB staining give less intense activity bands in zymogram, whereas bands which hardly appear after CBB staining give more intense activity bands after zymogramphy. So I wanted to ask is there any relation between protein concentration and activity band intensity in zymogram?? Thanks
Hi Gopal,
For my experiment, once I load more MMP protein into the well, the intensity of activity band is increasing after CBB staining. So, Intensity of activity band is related with your loading protein concentration. Hopefully, it can help you.
For the same protein, I would also expect to see more activity for more protein. If you are comparing two different proteins, then this might not be the case because one protein might have much greater activity than the other.
Thanks Tsung and Adam for your reply. I also expected the same, but zymogram is just opposite. I read about effect of enzyme concentration on enzyme activity it says that more enzyme increases the rate of reaction and comes to equilibrium faster than with less amount of protein (enzyme), could that be a possible reason?? In my case protein is same.
I don't have experience with zymography, but I can imagine a possible explanation in the stability of the product that is detected. If the larger amount of enzyme causes all the substrate to be converted to product rapidly, and the product then breaks down and becomes invisible (or diffuses away) before it can be observed, it might appear that there was less activity than when the smaller amount of enzyme converted the substrate to product more gradually. To test whether this is the case requires a kinetic approach - observe the formation of product over time.
Dear Gopal Tiwari
I agree with Adam and Valesca. In your case it is necessary to incubate the less time, just enough to just barely see the work of the enzyme in smaller amounts, the exhaustion of the substrate then is eliminated in the case of more enzyme. At the same time substrate concentration can be increased if a technique allows.
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