Hi,
I'm attempting to carry out an activity assay on a PDC enzyme using an indirect enzyme coupled assay. The reaction is started when PDC in cell extract (which has been heated to 60 degrees Celsius with TPP and MgCl2 for 30 minutes as directed by the protocol) is added to water, buffer at pH 6.0, ADH from yeast, NADH and pyruvate in acrylic cuvettes. At 340nm there should be a steady decrease in absorbance over the course of 5 minutes but having attempted the assay twice, my results are all over the place! Really high, really low and then high again! I don't know what I'm doing wrong as I'm following the protocol exactly and even on my second attempt, I ensured the contents of the cuvettes were thoroughly mixed before taking the absorbance as I though this may have caused a problem in the beginning! If anyone could shed some light on the situation that would be great!
Thank you in advance!
Try it in quartz cuvettes. Your plastic ones may have high absorbance at 340 nm. Check this by taking an absorbance spectrum of the empty cuvette against an air baseline. If that isn't the problem, make sure the absorbance of the assay solution is not too high for the instrument to read correctly.
After PDC is heated does the reaction mixture have room temperature?
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