Extraction Method： cold enzyme assay buffer [4℃,10 mM nicotinamide, 20 mM sodium bicarbonate, 100 mMsodium carbonate (Na2CO3), pH 11; a buffer classically usedfor enzymatic NAD(P)(H) analysis]; the same cold enzymeassay buffer with addition of detergent [0.05% Triton X-100and 1% dodecyl trimethylammonium bromide (DTAB)].
Please clarify: Are you saying that you are running the enzyme reaction to convert NADP+ to NADPH with G6PDG + glucose-6-phosphate at pH 11? I would not expect the enzyme to be active at such a high pH. A neutral pH would be more appropriate.
Also, you wrote that you use nicotinamide. Did you mean NADP+?
Adam B Shapiro My specific operation is as follows: use this to extract NADPH and NADP+ in cells or tissues to make tissue homogenate, then take 50 μl of tissue homogenate and add 100 μl of buffer, incubate for 10 minutes, and then add 10 μl of chromogenic solution. However, it is strange that the reaction seems to be inhibited using this extract.
The components of the buffer are：Tris-Hclbuffer ， Mg2+ ， G6P and G6PDH.
The components of the chromogenic solution are：1-MPMS and WST-8
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs