As the enzyme contains Mg2+ion bound in the native state, the buffer used for its assay also contains 10 mM MgSO4. I read somewhere that the buffer should contain EDTA and no reducing agent and must be degassed. Is it possible to use DTNB with this buffer? Is there an alternative to it if not?
Thanks
EDTA is not very important
(It is mainly the because transient metals can accelerate oxidation),
so it should work.
Just make sure you prepare a standard curve in the same buffer. Do not use any thiol-containing reducing agent (DTT, mercaptoethanol) that would react with DTNB.
Yes, Misha and Adam are correct. To add to Adam's point about the buffer: alkaline sample pH's can cause the yellow color to form and give higher background which then reduces assay sensitivity. Have you ever noticed how the DTNB stock solution turns slightly yellow when made by standard protocols or if stored at room temperature?
Thank you all for clearing the doubt.
@Adam. Are there any compatible reducing agents which can be used with ellman's reagent? I think TCEP also breaks DTNB to give TNB ions.
As you say, TCEP is not compatible with Ellman's reagent. Here is a reference about that:
http://www.ncbi.nlm.nih.gov/pubmed/7978256
I don't know of any reducing agent that you could use that would be compatible.
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