I am curious, because in existing papers i can't seem to find a method of analysis that explains the big fluctuations of absorbency I am experiencing. I should be observing a linear decrease or no decrease at all ,but i am also experiencing a rise in absorbency which is weird because i can't see de novo NADPH production account for it.
Thanks
P.S.: I am working in lysed cells.
Because of the UV wavelength (340 nm) of this assay, it is prone to interference due to turbidity. If there is even a little bit of precipitation during the assay, the absorbance will increase. You may not be able to see it with the eye.
If you have a dual-beam spectrophotometer, you can compensate for this problem using a sample in the blank beam that has the same composition, but leaves out one of the substrates or enzymes that you add to the the lysate. If you are using a plate reader, you can compensate by preparing blank wells the same way, and subtracting the blank well signal.
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