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NADPH monitoring


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6 replies to this topic

#1 cardosopedro

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Posted 22 April 2013 - 05:17 AM

Hello.

I would like to measure the levels of NADPH in my samples throughout time. I know there are some methods, but since I have no experience on this, I was wondering if someone could suggest me a good method. I work with a filamentous fungus (in case this is important to decide which is the best method). If possible I would like to do the assay on 96-well plates.


Thank you for the suggestions.
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#2 mdfenko

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Posted 23 April 2013 - 03:57 AM

take timed absorbance readings at 340nm.

increasing absorbance indicates increasing nadph.
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#3 cardosopedro

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Posted 23 April 2013 - 08:47 AM

How exactly? I should do total cell extracts first, right? With which buffer?

#4 mdfenko

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Posted 23 April 2013 - 10:37 AM

we were assaying specific enzymes from extracts and partially purified. and, we added nadp to the assay (as well as substrate and other components).

if you are just going to follow endogenous nadph then you may not want to do more than crack the cells open. you could use a medium that maintains intracellular conditions (ionic strength, pH, etc) but you will be diluting the extracts (which may not be a problem, if not too much and you know of the dilution factor).

we would need to know more about what you are doing to give any better advice.
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#5 cardosopedro

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Posted 23 April 2013 - 10:48 AM

I work with a filamentous fungus. I will treat the cells with a compound that I think will cause a 'metabolic shift' to the pentose phosphate pathway. Therefore, I expect alterations on the levels of NADPH. I want to do a kinetics experiment: treat the cells and measure NADPH during a time course. Could you please give me some references so I can check the procedure and buffers in detail?

#6 mdfenko

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Posted 23 April 2013 - 11:07 AM

i was simply performing assays of specific enzymes in the pentose phosphate pathway. i can give you some general references but they may not be what you're looking for.

glucose-6-phosphate dh assay (worthington)

here is a paper that came from the lab i worked in in school:

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#7 cardosopedro

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Posted 23 April 2013 - 11:23 AM

Thanks.




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