Im trying to measure the rate of enzyme activity based on the *oxidation* of the cofactor, NADH, measured by a decrease in absorbance at 340nm as NADH is converted to NAD+.
The buffer I am using is 10mM Hepes, 100mM NaCl, pH 7.5 @ 30'C.
Initially I am just trying to do a really basic titration of NADH from 0uM, upto 250uM to investigate the effect of NADH concentration on absorbance, using the assay buffer alone as a control.
However after measuring the absorbance, the assay buffer alone has a reading of ~0.6, as does the 250uM NADH sample.
I tried using distilled water, which also gave a similar reading. Does this seem plausible, water with an OD of 0.6? Or does it sound like a fault with the spectrophotometer?
>>I noticed in a older post someone mentioned having an absorbance with 500uM NADH of 1.04 after standardisation using water.
Any advice would be appreciated.
Edited by PatrickGuest, 01 November 2012 - 11:41 AM.