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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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  • $\begingroup$ What does DNSA stand for? Is this a kinetic assay or do you read the absorbance after a long period of time (like 30 min)? $\endgroup$ – LDC3 Aug 16 '14 at 0:36
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    $\begingroup$ DNSA is Dinitrosalicylic Acid Reagent which stops the reaction, I read the absorbance about 20 minutes after boiling $\endgroup$ – Oli Aug 16 '14 at 21:24
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Now I understand what your problem is. The dinitrosalicylic acid reagent reacts with glucose to give a colored reagent. It is best to read the absorbance quickly since the reagent becomes darker with time. So it is best to cool the tubes in a water bath.

I believe that your substrate contains free glucose which is why you get a high absorbance for your blank. You can either use the blank to zero the spectrophotometer (since you want the difference between the blank and the reacted solution), or you can find a different pectin which has less glucose in it.

Since there is some variability in the reaction, it is best to do everything in duplicate or triplicate (including the blanks).

This web page uses the wavelength of 575 nm. This might help with the intensity.

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  • $\begingroup$ But,what do you mean "You can either use the blank to zero the spectrophotometer" cause I calibrate the spectrophotometer using reagent / substrate blank ? $\endgroup$ – Oli Aug 17 '14 at 19:44
  • $\begingroup$ Usually, the zero on the spectrophotometer is set with water in the cell. What I mean, is that you have the blank solution in the cell and set the spectrophotometer to read an absorbance of 0 (or the transmission of 100%). $\endgroup$ – LDC3 Aug 17 '14 at 20:12
  • $\begingroup$ How long do I have to wait, to add DNSA reagent after incubating substrate with enzyme and after adding DNSA reagent how long do I have to wait for boiling ? $\endgroup$ – Oli Aug 19 '14 at 11:58
  • $\begingroup$ For the blank, you do not need to wait before adding DNSA reagent. I get all the reactions done (usually only two 3 min reactions and I make the blanks while waiting) then put all the tubes in the boiling water. The tubes are in the boiling water for 15 min. $\endgroup$ – LDC3 Aug 19 '14 at 13:32
  • $\begingroup$ Now,, the Enzyme blank values are better, ,, however, what range is assumed to be good in Pectinase Assay ? Mine Enzyme blank OD readings are b/n 250-500 $\endgroup$ – Oli Aug 21 '14 at 19:24

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