I've recently been trying to investigate the inhibition of H2S-synthesising enzymes: CBS, CSE and 3-MST, via PCR. We've ordered EGCG, PAG and i-3MT-3 to inhibit each enzyme respectively, and have tried multiple concentrations to determine the optimal concentration of the inhibitor needed to completely inhibit the enzyme without causing cell death.
For some of these compounds, we've used the maximum concentration possible which doesn't cause cell death and still can't achieve total inhibition (seen by the presence of bands in cells treated with the inhibitors in PCR).
It's been recently brought to our attention that the inhibition may only be pharmacological inhibition and not gene silencing, which may be why we can still see the gene sequence for the enzymes when we carry out PCR on the cells treated with the inhibitors.
So is there a way for us to test if the inhibitor is solely a pharmacological inhibitor and not a gene silencer?
If you have the capability to overexpress and purify these enzymes, or if you can purchase them, and have a method to measure their catalytic activity, you will be able to measure the effect of each compound on enzyme activity.
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