I am trying to measure the intracellular NAD levels in yeast using alcohol dehydrogenase assay. The reaction mixture includes Tris/Glycine (pH 9.5), ethanol, and extract prepared with 1M formic acid in 20% TCA or blank sample (just 20%TCA). Our ADH is a little old so I am using double the recommended amount otherwise the reactin takes too long (~20min).
However, once I add the ADH, the absorbance at 340nm increases gradually up to 1 or so even with the blank sample which supposedly does not include any NAD. Has anyone experienced something akin to this? Could there be NAD contamination in any of the solutions? I prepared Tris/Glycine solution from scratch using Sigma chemicals but not sure how the ADH enzyme was treated previously. Anything else that shows absorbance at 340nm? Any input is greatly appreciated.
Thanks!
By reading your post tow explanation came into my mind :
- first : do you centrifuge the ADH solution before adding ? To my experience, ADH is cryosensitive, ie start precipitate at T°
Thank you John and Eric for your valuable inputs.
The ADH I used is in (NH4)2SO4 solution kept @4oC and it does look turbid as you mentioned Eric. But if that is the culprit, wouldn't the absorbance increase be sudden once I add the ADH into the reaction mix? After I add the ADH to the reaction mix and thoroughly mix, the absorbance gradually increases and stabilizes by 15min.
This is a leftover ADH solution from another colleague (7 years past its expiration date), who knows what is in it! As John suggested I will order a fresh one and hope the problem goes away. Thanks again for your time!
http://www.sigmaaldrich.com/catalog/product/roche/10127558001
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