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#chromogenic substrate

2 public questions tagged with this topic.

Flow cytometry, FACS, ELISA

O-Phenyl diamine is used as a chromogenic substrate in?

ELISA relies on enzyme conjugated antibodies converting chromogenic substrates into colored products measurable spectrophotometrically. Horseradish peroxidase commonly uses TMB, ABTS and O phenylenediamine OPD producing orange brown soluble product read at 492 nm after acid stop. Alkaline phosphatase uses pNPP. OPD offers good sensitivity and stability for peroxidase detection. RIA uses radiolabeled isotopes measured by gamma counter, Southern and Western blotting may use chemiluminescence or colorimetric substrates but OPD is classic ELISA chromogen, not used in nucleic acid hybridization or radioactivity detection protocols.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Microscopy

Which chromogenic substrate gives purple product with ALP?

Alkaline phosphatase paired with nitroblue tetrazolium chloride chemistry generates insoluble formazan precipitate through reduction. NBT accepts electrons, converting from yellow soluble tetrazolium to dark purple-blue diformazan that deposits at enzymatic site. Often combined with 5-bromo-4-chloro-3-indolyl phosphate which yields indigo intermediate, enhancing localization. TMB is substrate for horseradish peroxidase, not alkaline phosphatase, giving blue. ONPG is hydrolyzed by beta-galactosidase to yellow ortho-nitrophenol. Para-nitrophenyl phosphate with phosphatase produces yellow soluble product, not purple. Thus NBT deposition indicates phosphatase activity in blotting and in situ assays.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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