8 replies to this topic

[PatrickGuest]

Hey all, I am trying to do a really basic assay but am having some difficulty.

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.

Patrick

Edited by PatrickGuest, 01 November 2012 - 11:41 AM.

[hobglobin]

But you have to use the buffer alone as blank and to reset the reading to zero with it, if the photometer isn't doing it automatically (modern photometers often have a blank measurement before the sample measurements and reset then automatically with blank)

[PatrickGuest]

Sorry I should have made that clearer, I did it both ways. I set the photometer to use the assay buffer as a blank, and then measure NADH absorbance once standardised against it which gave values in the region of 0.000 something, pretty much no difference, and then to ensure it was calculating it properly I ran the blank as a sample and manually calculated the difference (just subtracted the blank absorbance from the NADH treated sample) again with extremely small values.

The crux of the question is does a change in absorbance on the order of 0.000 seem likely between buffer alone and 250uM NADH, and does anyone know what the absorbance of distilled water is at 340nm?

That would tell me if the machine is working correctly.

Kind of specific but I hope someone knows.

Edited by PatrickGuest, 01 November 2012 - 11:07 AM.

[hobglobin]

it's long ago that I did such stuff, but in such a low range of readings you cannot expect linear relationships and the measurements can be quite arbitrary...
And I'm not sure about the range you use, but perhaps your concentrations are too low for the photometer (not really sure what the limits of detections are)?

[PatrickGuest]

Haha I should have made that clearer too, I ran 0 (assay buffer) 10uM, 50uM, 100uM, 150uM, 200uM, 250uM, and today I added 500uM.

The prior PhD student had been using similar concentrations and getting results, but didnt actually do the NADH titration, which makes me wonder if those conclusions are valid, as NADH absorbance is the measured variable and if this measurement may not be reliable (at least according to my experiements).

The sensitivity of the machine is something I hadn't thought of though, thanks I will check it tomorrow,.

[hobglobin]

And you use a suitable cuvette for 340 nm? And you can calculate with lambert-beer the concentrations and/or absorbance that you should get or have with the given dilutions.
And I'm pretty sure that the absorbance of distilled water will be near zero...surely some resins and stuff will absorb a bit but the water itself not, or almost not.

[PatrickGuest]

Its usually a Nunc 96 well plate. I didnt think of using the beer-lambert that way, I'll do that tomorrow.

Ill also run it with just the plate to see if thats absorbing.

Thanks, I'll update tomorrow how I get on.

[PatrickGuest]

Ran a new plate today containing empty wells, 160uL MilliQ water, 160uL Hepes Assay buffer, 160uL MilliQ with NADH @ 1mM, 160uL Assay buffer with 1mM NADH (all n=8 as it was convenient on the 96 well plate).

Absorbance is odd. Empty wells ~ 0.03 - as expected very low
Everything else ~ 0.45 - addition of 1mM NADH made no difference to the absorbance.

I then ran MilliQ water, assay buffer and assay buffer + 1mM NADH through an older spectrophotometer (using cuvettes). Absorbance @ 340nm:
MilliQ = 0.101
Hepes assay buffer: 0.103
Buffer + 1mM NADH: 0.204

So this photometer showed much lower absorbance for both water and buffer alone, and a marked increase with NADH.

Looks like theres something not right with the 96 well plate reader?

[hobglobin]

PatrickGuest, on 02 November 2012 - 10:19 AM, said:

Ran a new plate today containing empty wells, 160uL MilliQ water, 160uL Hepes Assay buffer, 160uL MilliQ with NADH @ 1mM, 160uL Assay buffer with 1mM NADH (all n=8 as it was convenient on the 96 well plate).

Absorbance is odd. Empty wells ~ 0.03 - as expected very low
Everything else ~ 0.45 - addition of 1mM NADH made no difference to the absorbance.

I then ran MilliQ water, assay buffer and assay buffer + 1mM NADH through an older spectrophotometer (using cuvettes). Absorbance @ 340nm:
MilliQ = 0.101
Hepes assay buffer: 0.103
Buffer + 1mM NADH: 0.204

So this photometer showed much lower absorbance for both water and buffer alone, and a marked increase with NADH.

Looks like theres something not right with the 96 well plate reader?

that's a possibility, did you check if the plates are UV transparent ?