Chromogenic products of the enzymatic reaction on the western blot membrane are visible to the eye and mostly stable for a long time. Chromogenic substrates for HRP include diaminobenzidine (DAB), tetramethylbenzidine (TMB), and azino-ethylbenzothiazoline-sulfonic acid (ABTS) salts. In the case of AP, the substrate is tetrazolium salts that are reduced to insoluble formazans. After incubation of the western blot membrane with a secondary antibody conjugated with AP or HRP, the membrane is incubated with a solution containing a substrate for the enzyme. Two of the most commonly used enzymes in colorimetric detection are alkaline phosphatase (AP) and horseradish peroxidase (HRP). This is the basis of colorimetric and chemiluminescent detections, respectively. The reaction results in formation of an insoluble colored precipitate on the membrane or release of photons that can be detected and quantified. The enzyme catalyzes conversion of a substrate, which is strictly localized to sites on the membrane where the secondary antibody bound to. Following incubation of the membrane with the secondary antibody, solution containing specific substrate is applied to the membrane. The secondary antibody provides binding specificity towards the target protein recognized by the primary antibody, while the attached enzyme is used for detection. One of the most common methods of detection in western blot is based on the use of a secondary antibody conjugated with an enzyme. Suggestions: Do not use, more contemporary methods are more effective – see below. Limited shelf-life of detection antibodies due to radioactive decay of the label.Dangerous – requires specific radiation safety training, appropriate personal protective equipment (PPE), registry and waste disposal systems.Quite laborious –secondary antibodies usually have to be labeled with radioisotopes by the end users.Once a popular technique, its use nowadays is relatively rare.Īdvantages: Radioactive detection offers good sensitivity and is very quantifiable. Iodine-125 was a popular choice with a relatively long half-life and emission of gamma rays consisting of low-energy photons that can be detected easily using X-ray films. One of the first methods of primary antibody detection to be developed was the use of secondary antibodies labeled with radioisotopes (PMID: 388439). Detection methods for western blot including radioactive, enzymatic (colorimetric & chemiluminescent), and fluorescent analysis Credit: ©Proteintech.
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