Cell lysis by alternate freezing and thawing--Deproteinization with 10%, 5--sulfosalicyclic acid, centrifugation-supernatant (50 micrlitre) + 1ml Phos.buffer + NADPH (50 microlitre, 4mg/ml) + Deoxynucleotide triphosphate (20 micro.l, 1.5mg) + GSH reductase (6 units/ml)--mix--5 min observation in UV at 412 nm.
In this method why dNTPs are employed?, What is their role? and how to use it?
Dear valued
Niranjana Kamalapriya, Please find attached files.
Regards
Ali
Hi, I suppose you meant DTNB.
The assay measures GSH and GSSG after its reduction to two
molecules of GSH (2GSH) by Glutathione reductase in the presence of NADPH. GSH reacts with DTNB to form TNB and GS-TNB (glutathione adduct of TNB). GS-TNB is then reduced by GSH with the formation of GSSG and TNB or (more probably)
by GR and NADPH with the generation of GSH and TNB. The formed TNB
is measured at 412 nm.
Hi Niranjana,
when GSH reductase is used, you measure the total GSH in cells, ie, GSSG is reduced to GSH.
Dear Ali,
Thank you for those papers. They were very helpful.
Dear Ranieri Rossi,
If you could please check the link I've shared, it wld be clear what I'm asking for.!. In that paper they have mentioned deoxynucleotide triphosphate, and I couldn't get the principle behind it., and thank you for responding.
Dear Shimin Liu,
Yeah I get the GSH, GSSG, DTNB concept., but my doubt is actually based on the paper I've shared here. Thank you for explaining though.
Hi Niranjana
I send you a file for the original method, which may help you to understand the principle better.
Dear Niranjana,
the use of deoxynucleotide triphosphate is just an error probably due to a flawed copy-paste. As matter of fact it is not reported in the original procedure form Akerboom and Sies (Meth Enzymol ref #17 in the paper you shered). Do not consider it. You can follow the Tietze's original method as modified in the NP paper I have attached.
The method suggesteb by Shimin is quite different. It is an DTNB endpoint procedure, which is excellent when samples are abundant, not in the case of cell cultures where you have few sample. In this case the Tietze's recycling method is recommended.
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