Chromogenic substrates are utilized in colorimetric detection. They are easy and straightforward to make use of. Suitable for most immunotechniques – from immunohistochemistry to Western blotting and ELISA, they provide a really cost-effective methodology of detection.
Chromogenic substrates can be utilized in plenty of immunohistochemical purposes from staining tissue with IHC by way of to Western blotting. The chromogenic substrate is added to a blot or tissue beforehand incubated with an enzyme-conjugated antibody (usually horseradish peroxidase (HRP) or alkaline phosphatase (AP)), which converts the substrate to a coloured precipitate. Signal growth is arrested by merely washing off the substrate. The precipitate is seen to the bare eye, and on a Western blot, it may be detected with out particular gear as a coloured band. The chromogenic precipitate may be seen on the tissue below a standard mild microscope when used for immunohistochemistry (IHC). Colorimetric detection is simple to make use of, though it might require optimization or further staining to enhance sign over background in samples expressing low antigen.
Solubility
Chromogenic reagents can be found for the totally different reporter enzymes, HRP, AP, GOD (Glucose oxidase) and β-Gal (β galactosidase). The substrates can be found in numerous colours and sensitivities. Depending on the assay, a soluble or insoluble substrate may be chosen.
Alcohol insoluble chromogens are acceptable for Western blotting and IHC the place counterstains or mounting media are used. The soluble dyes are higher suited to ELISA and plate based mostly assays.
Western blotting and dot blotting
When utilized in Western blotting or dot blotting, the chromogenic substrate seems as coloured band or spot on the membrane on the areas of the immobilized antigen-antibody complexes (Figures 2 & 3). The blot may be developed till the sign of the bands attain the depth required, this permits for an excellent degree of sensitivity as the event of the blot may be noticed instantly and the response halted instantly by washing off the substrate. The developed membrane can then be imaged or saved for subsequent use. Samples with low pattern focus (low antigen) could require prolonged growth, which can result in larger background, obscuring bands.
ELISA
Colorimetric ELISA measures the optical density of a soluble coloured product which is often proportional to the quantity of analyte current within the pattern, permitting relative quantification of the analyte. Like different immunotechniques, ELISA requires optimization of timing and growth situations to acquire correct and reproducible assays.
Immunohistochemistry
Colorimetric detection generally is a very delicate methodology for the immunostaining of tissue and cells. Different substrates provide totally different sensitivities and colours to help identification, a number of antigen recognition is feasible as colours may be distinguished from one substrate and one other. Quite a lot of counterstains can be found which can be utilized to extend distinction in order that the IHC staining seems stronger. Figure 5 exhibits the brown colour of 3,3’ diaminobenzidine (DAB staining) together with hematoxylin counter-staining of nuclei.
Reporter Enzymes (conjugated to antibody)
The mostly conjugated reporter enzymes are horseradish peroxidase (HRP) from the horseradish plant Armoracia rusticana, and alkaline phosphatase (AP) from calf intestines, though Glucose oxidase (GOD) and ß-D-Galactosidase from E.coli will also be used. HRP and AP each provide totally different advantages relying on the appliance necessities. HRP is a small 40 kDa molecule which often binds to an antibody in a ratio of 4:1. Its small dimension means good intracellular penetration and it’s much less more likely to trigger steric hindrance with the antigen/antibody complicated. AP is barely bigger (86 kDa) which can trigger steric interference however the substrate-converting response is linear, which can imply the detection incubation might be prolonged and probably be extra delicate. AP additionally removes the potential for non-specific staining of tissues with excessive ranges of endogenous peroxidases – a limiting think about HRP.
Chromogenic substrates
Each reporter enzyme can be utilized with plenty of totally different chromogenic substrates which can be found in quite a lot of specs and codecs providing totally different sensitivities, some frequent ones are summarized within the desk beneath.
| Enzyme | Solubility | Substrate | Precipitate colour | Sensitivity |
| AP | Insoluble | BCIP & NBT (5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) & Nitro blue Tetrazolium) | Blue/purple | High |
| AP | Soluble | Fast Red TR (4-Chloro-2-methyl benzenediazonium) | Red/orange | High |
| HRP | Insoluble | DAB (3,3’-Diaminobenzidine) | Brown | Medium |
| HRP | Soluble1 | TMB (3,3′, 5,5;-tetramethylbenzidine) | Dark blue | High |
| HRP | Soluble | CN (4-chloro-1-naphthol) | Blue purple | Low |
| HRP | Insoluble | CN/DAB | Black | High |
| HRP | Soluble | ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) | Green | Low |
| HRP | Soluble | OPD (o-phenylenediamine dihydrochloride) | Yellow | Medium |
| AP | Soluble | PNPP (p-Nitrophenyl Phosphate) | Yellow | High |
| β -Gal | Insoluble | X-Gal (5-Bromo-4-Chloro-3-indolyl-B-D-Galactopyranoside) | Blue | – |
| GOD | Low solubility | t-NBT (2,2′,5-5′-tetra-p-nitrophenyl-3,3′ -(3,3′ -dimethoxy-4,4′-biphenylene)-di tetrazolium chloride) | Black/Brown | High |
