![]() ![]() 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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