1/24/13 Chromogenic Western Blotting Substrates

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Chromogenic Western Blotting

Chromogenic or precipitating substrates have been used widely for manyyears and offer the simplest and most cost-effective method of detection.When these substrates come in contact with the appropriate enzyme, theyare converted to insoluble, colored products that precipitate onto themembrane. The resulting colored band or spot requires no special equipmentfor processing or visualizing. Chromogenic blotting substrates are available ina variety specifications and formats. The appropriate substrate choicedepends on the enzyme label, desired sensitivity and form of signal ormethod of detection needed.

Introduction

Unlike chemiluminscent or fluorescent blotting applications, chromogenicsubstrates do not require special equipment for visualization of the assayresults. Similar to developing film, the blot is incubated in substate until thedesired amount of development. Unlike chemiluminescent blotting assays,the colored precipitate can not be easily be stripped off reprobing. Therefore,it is imporant to allow the reaction to process until color development issatisfactory and then stop the reaction.

The low sensitivity of chromogenic substrates makes it difficult to optimizethem for detecting proteins of low abundance. Though the reaction can beallowed to develop for several hours or even overnight, this also allowsbackground signal to develop as well. Where chromogenic substrates fail interms of sensitivity, they are ideal for applications where protein abundanceis high. Because the product of the substrate reaction is a coloredprecipitate, the signal is stable, therefore chromogenic substrates do nottypically have issues with false negative results (ghost bands) that can occurwith chemiluminescent substrates. Since chromogenic substates aretypically used to detect abundant proteins and reaction development can bemonitored visually, this gives them greater flexibilty for optimizationcompared to chemiluminescent or fluorescent blotting systems.

The performance of a particular substrate may vary dramatically whenobtained from different suppliers. This is because performance can beaffected by the concentration and purity of the substrate and by otheradditives and buffer components that are a part of the formulation.

Learn more...Overview of Western Blotting

Download Documents...Tech Tip #32: Guide to enzymesubstrates for Western blotting.

Order Literature...Western Blotting Handbook

Chromogenic Horseradish Peroxidase Western Blot Substrates

Peroxide must be added to a substrate for colorimetric detection with HRP.Because of its extremely short shelf life at the desired concentration,hydrogen peroxide traditionally was added to a buffer, along with thesubstrate, immediately prior to use. As a result, these substrates typicallyhave a useful shelf life of only a few hours. Many commercially availableprecipitating HRP substrates are supplied with, or come prepared in, stableperoxidase substrate buffer. The stablized peroxide in these solutions aregenerally concentrated and is less corrosive than the traditional 30% stocksolution of hydrogen peroxide. Since 30% hydrogen peroxide and dilutionsolutions of hydrogen peroxide are not stable, reagents prepared withstabilized peroxide will provide more consistent results.

Chromogenic substrates for Western blotting with HRP.

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1/24/13 Chromogenic Western Blotting Substrates

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Chromogenic substrates for Western blotting with HRP.

TMB, with a molecular weight of 240.4, is most often used as a substrate forHRP in ELISAs. However, in the presence of HRP and peroxide, a water-soluble blue product is generated that can be precipitated onto a membrane.1-Step TMB – Blotting is a single-component peroxidase substrate forWestern blotting and immunohistochemistry. Precipitating the productresults in dark blue bands where the enzyme is located. 1-Step TMB –Blotting is well suited to applications that require a large signal-to-noise ratio.

4-CN has a molecular weight of 178.6 and can be used for chromogenicdetection of HRP in blotting and histochemistry. This precipitate is not assensitive or as stable as TMB and DAB, but the alcohol-soluble precipitatephotographs well and has a distinct blue-purple color that can be useful indouble-staining applications.

DAB has a molecular weight of 214.1 and yields a brown precipitate in thepresence of HRP and peroxide. The brown, insoluble product can be readilychelated with osmium tetroxide. This property makes DAB ideal for electronmicroscopy. The color produced by DAB can be intensified with the additionof metals such as nickel, copper, silver and cobalt that form complexes. Thecolor produced by the metal complexes is darker than the color produced byDAB alone, enhancing the sensitivity in staining applications.

The individual benefits of 4-CN and DAB are often combined into a singlesubstrate mixture, CN/DAB Substrate. The CN/DAB Substrate has excellentsensitivity, yielding a dark black precipitate that photographs well. TheCN/DAB Substrate works well in Western blotting and dot blottingapplications.

View Products...ChloronaptholCN/DAB Substrate kitPrecipitating TMB Substrate

Chromogenic Alkaline Phosphatase Substrates for Western Blotting

NBT, with a molecular weight of 817.6, is a member of a class ofheterocyclic organic compounds known as tetrazolium salts. Upon reduction,the compound yields NBT-formazan, a highly colored, water-insolubleproduct. The substrate is widely used for immunochemical assays andtechniques because the color produced by the formazan is linear and stableover a wide dynamic range.

BCIP has a molecular weight of 433.6, and hydrolysis by alkalinephosphatase results in a bluepurple precipitate that can be deposited onnitrocellulose or nylon membranes. BCIP can be used as a chromogenicsubstrate for both immunoblotting and immunohistochemical studies.

An ideal system for blotting or staining applications with AP is thecombination of NBT and BCIP. Together, they yield an intense, black-purpleprecipitate that provides much greater sensitivity than either substrate alone.This reaction proceeds at a steady rate, allowing accurate control of itsrelative sensitivity. NBT/BCIP characteristically produces sharp bandresolution with little background staining of the membrane.

Fast Red TR/AS-MX Substrate (structure pictured below) is a sensitivesubstrate that results in a bright red precipitate on transfer membranes. Weno longer offer this particular substrate.

View Products...NBT/BCIP SubstratesFilter paper and membranes

NBT/BCIP reaction scheme. BCIP is hydrolyzed by Alkaline Phosphatase to form an intermediate thatundergoes dimerization to produce an indigo dye. The NBT is reduced to the NBT-formazan by the tworeducing equivalents generated by the dimerization.

1/24/13 Chromogenic Western Blotting Substrates

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Chromogenic substrates for Western blotting with Alkaline Phosphatase.

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