![]() ![]() Therefore, when using this enzyme for visualization, the membrane must be thoroughly washed to remove sodium azide. HRP activity can be inhibited by the presence of sodium azide and contaminants from low-grade glycerol. Although colorimetric detection cannot provide reliable quantitative data, blots developed under the same conditions/timings may be compared qualitatively. The developed membrane can be imaged using a CCD camera that enables the bands’ intensity to be analyzed using software such as Image J. However, this may lead to unacceptable background signal. Poorly expressed proteins may require prolonged incubations with the substrate of (up to) several hours or even overnight to produce a signal with suitable intensity. Less target protein results in fewer antibody complexes binding and consequently less conjugate. The speed of signal development is dependent on the abundance of the reporter enzyme to catalyze the reaction, which is governed by the abundance of the target protein and the primary antibody bound to it. Color development is monitored visually until it reaches satisfactory strength, following which the reaction may be stopped by washing excess substrate from the membrane or by applying a stop buffer to halt the reaction. The sensitivity of the substrate and the length of incubation determines the intensity of colored precipitate deposited onto the membrane. Modern formulations offer substrates in stabilized peroxide, which can provide more consistent results in a ready-to-use application. Traditionally, hydrogen peroxide is added to the buffer prior to use, giving the substrates a shelf-life of only a few hours. It is necessary to add Peroxide to the substrate when using HRP conjugates. Much higher sensitivities can be achieved using NBT/BCIP for AP and 4CN/DAB for HRP as combination substrates. Each substrate has a different affinity for its respective enzyme. Colorimetric Substrates for Horseradish PeroxidaseĬommonly used substrates for HRP include 3,3′, 5,5′-tetramethylbenzidine (TMB), 3,3-diaminobenzidine (DAB) and DAB in combination with 4-chloro-1-naphthol (4CN), which appear as dark blue, brown and black precipitates, respectively. Nitro blue tetrazolium (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) are two AP substrates used in combination with colorimetric detection to give a blue-purple precipitate. Colorimetric Substrates for Alkaline Phosphatase The enzyme reacts with the substrate, forming a detectable colored product. The substrate is added to the antibody-antigen complex.Ĭ. Enzyme-conjugated secondary antibody binds the primary antibody at the protein of interest.ī. The membrane is then “set” and can be analyzed by eye without imaging equipment. The bands are developed until the desired color intensity is reached before a stop solution is added to “quench” the reaction and prevent oversaturation, which may prevent result interpretation. The reaction can take a few minutes for a precipitate to develop. The substrates are converted to colored, insoluble products, which appear as colored spots or “bands” where the target molecule: antibody complex are captured on the membrane. The membrane is incubated with a reporter enzyme-conjugated secondary antibody, either Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP), after which a colorimetric substrate is added to the blot. Fluorescent western blotting uses a fluorescent dye molecule to provide a signal which is captured using an imager and may be used to facilitate the detection of multiple target proteins simultaneously.Ĭolorimetric detection offers fast results and is simple to perform, making it an attractive option for many researchers. Chemiluminescent substrates emit light, which may be visualized using film or a CCD camera. Colorimetric detection enables quick and straightforward visualization of the protein of interest without the need for expensive instruments.Ĭolorimetric substrates yield a colored product that can be viewed by the eye or quantified using a camera and detection software. The enzymes catalyze reactions with their substrates to generate products that can be detected. Although radioactive and colorimetric detection has been used historically, chemiluminescent and fluorescent detection have become increasingly popular.Ĭolorimetric and chemiluminescent detection utilize reporter enzyme conjugates. Target proteins can be visualized using a variety of detection methods. Detecting Multiple Target Proteins on the Same Blot – Multiplex Detection.Detecting Multiple Proteins with Chemiluminescent Detection.Substrates for Chemiluminescent Detection. ![]()
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