The DTNB assay Sample Clauses

The DTNB assay. The results described in chapters 3 and 4 relied on accurate measurements of intracellular thiol concentration. First described by Xxxxxx in 1959, 5,5’-dithiobis- (2-nitrobenzoic acid) (DTNB) or otherwise known as Xxxxxx’x reagent is the most widely used chemical for detection of thiol content150. Reduced thiols react with DTNB to cleave the disulphide bond and generate 2-nitro-5-thiobenzoate (NTB-). In water, NTB- becomes an NTB2- ion which is yellow in colour. The change in colour associated with DTNB conversion to NTB2- is therefore directly proportionally to thiol concentration. To measure thiol content with the DTNB assay, 150 µL DTNB (Sigma-Xxxxxxx, UK) was added to samples pipetted into clear microtitre plate. The plate was incubated for 5 mins before 50 µL of NADPH solution was added to each well and mixed with a pipette. A spectrophotometer plate reader was then used to measure the absorbance of each well at 412 nm.
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