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.Traces of ß-mercaptoethanol will damage the antibodies.Rinse the membrane under running water tap for 1–2 min.Dispose of the solution as required for ß-mercaptoethanol based buffers.Incubate at 50☌ for up to 45 min with some agitation Add the buffer to a small plastic box which has a tight lid use a volume that will cover the membrane.Prepare buffer and strip membranes under a fume hood.Īdd 0.8 mL ß-mercaptoethanol under the fume hood Repeat incubation for 5–10 min with fresh stripping buffer.Using a volume that will cover the membrane, incubate at room temperature for 5–10 min.If stripping is judged to be satisfactory, rinse the membrane several times with buffer, then block before proceeding to the antibody incubation.īring volume up to 1 L with distilled water Some researchers report successfully staining a membrane after stripping ten or more times.Įfficiency of stripping can be checked by incubating the membrane with chemiluminescent detection reagent. These steps can be repeated for probing with several antibodies, though the potential signal may be weaker and the background higher after each round of stripping. As a rule of thumb, try the gentler one first and then proceed to the harsher one if there is still a signal from the antibody that one is trying to strip. The following two protocols differ in harshness of treatment. Chemiluminescent reagents such as ECL are recommended as they will not leave a stain and are more sensitive than colorimetric reagents. Note also that colorimetric/chromogenic detection reagents will leave a permanent visible stain on the membrane that can interfere with subsequent detection of targets of similar molecular weights. For the same reason, a stripped membrane should not be probed to demonstrate the absence of a protein.Ī PVDF membrane is highly recommended to minimize loss of sample protein. It is not advisable to make quantitative comparisons of targets probed before and after stripping since the procedure removes some sample protein from the membrane. When probing for multiple targets, stripping and re-probing a single membrane instead of running and blotting multiple gels has the advantage of saving samples, materials, and time. Stripping is useful when one wants to investigate more than one protein on the same blot, for instance a protein of interest and a loading control. Stripping is the term used to describe the removal of primary and secondary antibodies from a western blot membrane.
Our protocol for western blot membrane stripping and restaining includes step-by-step details on the removal of antibodies from western blot membranes.
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