I am gonna do an Ellman assay and based on the datasheet I should measure the molar absorption at 412 nm. I wonder ELISA reader or nanodrop one can be used to do measurements at this wavelength or not.
Especially the amount of sample volume in the cuvette over the spectrophotometer is too much.
The assay should work fine on those platforms. But instead of calculating directly from the Beer-Lambert extinction coefficient, I recommend you run a standard curve with each assay set. That will give you some method validation and statistical power.
Note you don't need to worry about variable sample dilution in a plate reader as dilution is directly proportional to path length, so the effects cancel each other.
Dear Dr. John Carter Thank you so much for your reply. I will do that. shall I prepare Elman reagent for every assay freshly? Is it not possible to prepare it like Bradford reagent and keep refrigerated?
I never tried to keep Ellman or Bradford overnight. I also never tried doing them in 96-well plates, so I understand why you're asking. You can try. You'll probably see increased background, which you may find you can simply subtract. And that will come with decreased dynamic range, which lowers your assay "ceiling". The reagents are not terribly expensive, so if you have time, you can play around with storage.
Yes, there are a number of commercially available kits based on DTNB. The reagent is usually prepared at a fairly high concentration so even if a small amount is converted to the thiol form, it gives a pretty small increase in background. As was mentioned, use a standard curve of known amounts of thiol to calibrate your assay response. It should be useful for up to around 100 nanomoles of thiol. At that point absorbance is approaching 2 and you could start seeing nonlinear effects.
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