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I am working on pectinase enzyme assay. I incubated 900 ul of substrate for 10 minutes in the water bath, followed by adding 2ml of DNSA reagent, then 100ul of enzyme extract added finally i read the absorbance @ 540 OD. However the values are high. How can I troubleshoot high enzyme blank values in Pectinase assay ?

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  • $\begingroup$ So you have the problem that your blank is too high? $\endgroup$ – Chris Aug 16 '14 at 22:06
  • $\begingroup$ Yeah, the enzyme blank is high $\endgroup$ – Oli Aug 16 '14 at 22:11
  • $\begingroup$ Can you exclude having a contamination? $\endgroup$ – Chris Aug 16 '14 at 22:11
  • $\begingroup$ What contaminants ? As much as possible the enzyme production is done in contamination free environment. $\endgroup$ – Oli Aug 16 '14 at 22:15
  • $\begingroup$ You are not doing this assay in your hand, are you? So what about you reaction tubes, the photometer cuvette etc.? $\endgroup$ – Chris Aug 17 '14 at 10:26
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So, your blank is everything but the enzyme? That is, substrate and DNSA with 100 uL water? If yes, measure, as negative controls, substrate alone and DNSA alone. If one of them is also highly absorbing light, it may be contaminated, and a new vial should be ordered. If both are highly absorbing, it may be the water you are using, or the spectrophotometer.

Alternatively, you may try to measure a time course. If blank's absorbance is increasing as time goes by, there is contamination, and reordering reagents may help you. If absorbance stays constant, you are not looking at an enzyme contamination, that is, the culprit is spoilt water, or broken spec, or even wrong protocol. (That is, reordering or repeating won't help.)

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