To calculate the enzyme activity, we have used Rhodanin assay (Shweta et al., 1999). But at the end of the assay the results are not coming as earlier reported. There is formation of chromogen in both the test tubes i.e. experimental and control. There is no contamination in the (Sigma Mol Bio grade used) chemicals.
Please tell what could be the possible reason of this outcome.
Thanks in advance.
Hi there,
The problem is the control experiment, I guess... How do you run this control assay and what is the actual composition of the mix?
Do you mean there is no difference in read OD for experiment and control assays?
Yes, there is no difference in OD for experiment and control.
In control, the composition is substrate solution (i.e. 0.01M Methyl Galate in 0.05M citrate buffer, pH-5.0), then 0.3ml methanolic rhodanine, then 0.2 ml KOH.
And 0.25ml enzyme extract added at the end for experimental one.
Hopefully this will help to find out the reason.
Do you follow the tannase assay described this paper:
http://ac.els-cdn.com/S0003269799944056/1-s2.0-S0003269799944056-main.pdf?_tid=1623efb6-0150-11e6-be78-00000aab0f26&acdnat=1460535601_a1edd0d977d22b82daff7778c2b240f9
If yes, enzyme extract is added at the very end only for the control assay. In the other assays, enzyme is added for the first princubation.
If you follow the right protocol then the problem is either the reagents and/or the enzyme solution...
Yes, enzyme extract was added at the end and reagents were of good quality. Thank you so much for your help.
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