Both Ponceau S staining and the molecular weight markers may be used to cut the membrane after transfer for probing the resulting parts with different antibodies.
Technical Note : This immunodetection procedure is provided as a guideline only. Optimization may be required for each antibody; specific information can be found in most data-sheets of the commercially available antibodies e. During this last process, the target protein will be detected using a specific antibody and will appear as a band on the film.
The position of the band is dependent of the molecular weight of the target protein, whereas the band intensity depends on the amount of target protein present. Blocking: To avoid non-specific interactions of the antibody with the membrane the excess space on the membrane is covered with a dilute solution of a generic protein.
Probing: The protein of interest is detected by a specific primary antibody. After the unbound primary antibody is washed away, the membrane is exposed to a different antibody linked to the reporter enzyme , horseradish peroxidase HRP.
This secondary antibody is directed against the species-specific portion of the primary antibody e. Detection: A chemiluminescent agent is used as a substrate that will luminesce when exposed to the HRP on the secondary antibody. This reaction produces luminescence in place and in proportion to the amount of probed protein.
The light is then detected by photographic film. A generic step-by-step procedure is provided below. Other detailed procedures can be found in the ECL Plus Western Blotting Detection Reagents instruction manual 5 or in most data-sheet accompanying the commercially available antibodies.
Incubation time, antibody dilution, and blocking and wash solutions have to be empirically optimized for each antibody. Incubate the membrane with your primary antibody specific for the protein of interest. The primary antibody should be diluted in blocking solution. For most antibodies, complete binding is achieved after hours incubation at room temperature under gentle agitation. Then 3X10 minutes with PBS, 0.
Incubate the membrane with a HRP-coupled secondary antibody diluted in blocking solution. Choose an antibody which recognizes the IgG portion of the species where the primary antibody was raised.
The incubation is performed for 1 hour at room temperature under gentle agitation. Discard the solution and quickly wash the membrane once. Proceed to the chemiluminescent detection according to the manufacturer instruction. Place your membrane in saran wrap or a pouch and place inside an autoradiography cassette.
Make sure to drain any excess liquid and remove all air bubbles. Expose the photographic film to membrane in a dark room. Start with a 1 minute exposure time and adjust according to desired signal intensity.
Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein. We recommend using TBS if you plan to probe your membrane with phosphospecific antibody.
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