AP is slightly larger (86 kDa) which may cause steric interference but the substrate-converting reaction is linear, which may mean the detection incubation could be extended and potentially be more sensitive. Its small size means good intracellular penetration and it’s less likely to cause steric hindrance with the antigen/antibody complex. HRP is a small 40 kDa molecule which usually binds to an antibody in a ratio of 4:1. HRP and AP both offer different benefits depending on the application requirements. The most commonly conjugated reporter enzymes are horseradish peroxidase (HRP) from the horseradish plant Armoracia rusticana, and alkaline phosphatase (AP) from calf intestines, although Glucose oxidase (GOD) and ß-D-Galactosidase from E.coli can also be used. Reporter Enzymes (conjugated to antibody) Nuclei were counter-stained with hematoxylin. (B) GFP expressing cells were visualized by brown staining in the positive control Signal was visualized using the chromogenic substrate 3,3’ diaminobenzidine (DAB) substrate (Envision kit DakoCytomation, Glostrup, Denmark) for 10 min at room temperature. Samples with low sample concentration (low antigen) may require extended development, which may lead to higher background, obscuring bands.įigure 5: Anti-GFP immunohistochemical staining using Biotin-SP conjugated Donkey anti-Rabbit IgG (H+L) Secondary antibody followed by HRP streptavidin: (A) At 4 weeks post-transplantation, no GFP signal could be detected in the in vivo specimens. The developed membrane can then be imaged or stored for subsequent use. The blot can be developed until the signal of the bands reach the intensity required, this allows for a good level of sensitivity as the development of the blot can be observed directly and the reaction halted immediately by washing off the substrate. When used in Western blotting or dot blotting, the chromogenic substrate appears as colored band or spot on the membrane at the locations of the immobilized antigen-antibody complexes (Figures 2 & 3). The soluble dyes are better suited to ELISA and plate based assays. Depending on the assay, a soluble or insoluble substrate can be chosen.Īlcohol insoluble chromogens are appropriate for Western blotting and IHC where counterstains or mounting media are used. The substrates are available in different colors and sensitivities. The reporter enzyme conjugate catalyzes the conversion of the chromogenic substrate to a colored insoluble precipitate, visible by eye on the blotting membrane.Ĭhromogenic reagents are available for the different reporter enzymes, HRP, AP, GOD (Glucose oxidase) and β-Gal (β galactosidase). Chromogenic substrate is added to the antibody-antigen complex. Reporter enzyme-conjugated secondary antibody detects the primary antibody for the protein of interest. Colorimetric detection is easy to use, although it may require optimization or additional staining to improve signal over background in samples expressing low antigen.įigure 1: Indirect colorimetric detection. The chromogenic precipitate can be seen on the tissue under a normal light microscope when used for immunohistochemistry (IHC). The precipitate is visible to the naked eye, and on a Western blot, it can be detected without special equipment as a colored band. Signal development is arrested by simply washing off the substrate.
The chromogenic substrate is added to a blot or tissue previously incubated with an enzyme-conjugated antibody (typically horseradish peroxidase (HRP) or alkaline phosphatase (AP)), which converts the substrate to a colored precipitate.
Suitable for most immunotechniques – from immunohistochemistry to Western blotting and ELISA, they offer a very cost-effective method of detection.Ĭhromogenic substrates can be used in a number of immunohistochemical applications from staining tissue with IHC through to Western blotting.
#Dot blot 96 wells pdf#
Download PDF Chromogenic substrates are used in colorimetric detection.
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