In my assay, the substrate contributes a lot of DMSO, about 3.2% at a 100uM and contributes 0.8% making a total of 4% DMSO concentration in the assay. So we decided to make the DMSO concentration 4% in all the wells. I know it is recommended not to go above 2% but we need higher substrate concentration because the Michaelis-Menten curve did not saturate.However in another assay where the DMSO concentration is not constant across the wells (about 3.6% is highest in one well and 2% the lowest) curves are in the expected order. The percent inhibition also appears to have decreased.
When you saw that the activity was higher with 2 µM inhibitor than with none, did both have the same DMSO concentration, or was there a difference? How does the enzyme activity vary with DMSO concentration? How much higher (%) was the activity at 2 µM inhibitor?
Hi Adam, both the 2uM and no inhibitor are kept at the same DMSO concentration (4%). The 2uM is about 10-20% higher than the zero.
Thank you
Also there appears to be no difference in activity between the two different DMSO concentrations for the zero its just that the 2uM appears to have increased in activity with increasing DMSO concentration
Do you consider the 10-20% difference to be statistically significant, or could it just be experimental variation? Are you using cuvettes or microplates? Sometimes there can be positional variations in activity in microplates due to temperature differences across the plate.
In addition to ruling out DMSO as a factor, don't forget to run a negative control containing inhibitor but no substrate, to make sure that the inhibitor solution itself, is not giving you an increased background signal.
My next suspect would be natural variance, especially from pipetting, especially if using a plate-based spectroscopy method as path length is now an extra variable. As Adam has pointed out, ensuring statistical reproducibility of the activation would be my next step, especially using fresh stocks, as compounds can change when stored in DMSO (things tend to get oxidised), even when stored at -80 C. Having ruled out any possible artifacts or errors, an inhibitor now displaying activation kinetics likely has a contaminant, perhaps a degradation product. LCMS, NMR, etc. would be your friend to find out if that is the case.
Thank you both very much. I am looking into the dilution of the inhibitor, could it be that there is less inhibitor than expected in the 2uM? the stock is diluted in 50% DMSO. So the 10-20% may be experimental variation and it may actually look like the zero? If so I might need to prepare new dilutions of inhibitor and see how it turns out.
Thank you.
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