b'Post-detection and signal enhancement Stripping and reprobing western blots protein is inevitably lost from the membrane, making it important to minimize this loss by stripping the antibody One of the major advantages offered by chemiluminescentunder gentle conditions. Because each antibodytarget and fluorescent detection methods is the ability to strippair has unique characteristics, there is no guaranteed reagents from the blot and then reprobe the samemethod to remove every antibody while preserving blot under modified or new conditions. This is a usefulthe target protein.technique when optimizing antibody concentrations, signal-to-noise ratios, or when multiple detection experimentsTesting and reprobing stripped blotsare to be performed on the same blot (Table 25). WithAfter any stripping procedure, test the blot to ensure that all chemiluminescence and fluorescence, all of the reagentsof the detection reagents have been removed. To do this, can be removed (stripped) from the membrane, in contrastwash the membrane several times, block, incubate with to chromogenic detection where a colored precipitate issecondary antibody, and then reincubate with the detection deposited on the membrane. substrate. If the primary antibody was effectively removed by the stripping procedure, no secondary antibody should Stripping western blot membranes bind to the membrane and no signal should be produced. The key to stripping a membrane is to use conditionsIf bands are still visible on the blot, the stripping conditions that allow the release of antibody from the target protein,must be intensified. Often a simple increase of the reaction without releasing a significant amount of protein fromtime or temperature will complete the stripping process. the membrane. Various protocols have been developedHowever, it may be necessary to alter the composition to accomplish this purpose, and they generally includeof the stripping buffer or change methods. A variety of some combination of detergent, reducing agent, heat, andThermo Scientific Restore stripping buffers are available low pH. During the stripping procedure, some amount ofto make the optimization process easier (Table 26).Table 25. Advantages to restripping and reprobing western blots.Conserves sample When the protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be reanalyzed with the same or different antibodies.Saves time It is time-consuming to run an SDS-polyacrylamide gel and then transfer the proteins to a membrane. By using the same blot for several different detections, you save time.Saves money By reusing the same blot, you save money on the costs of membrane, buffers, and protein sample.Assay optimizationThe signal intensity of high-sensitivity chemiluminescent substrates often requires antibody concentration optimization to is easier achieve the highest-quality blot. Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration.Quickly confirmWhen immunoblot results are not as expected, reprobing allows the use of the same protein sample without going back atypical results to gel electrophoresis to run new samples.Correct mistakes Immunoblotting requires many steps, providing ample opportunity for mistakes to occur. By stripping the membrane, the blot can be reused.Table 26. Stripping buffer selection guide.Restore Western BlotRestore PLUS Western BlotRestore Fluorescent Western Blot Stripping Buffer Stripping Buffer Stripping BufferFeatures Gentle, odor-free Robust yet gentle, odor-free Optimized for NIR fluorescent blotting Ready to use Yes Yes YesMembraneNitrocellulose and PVDF Nitrocellulose and PVDF Use with low-fluorescence PVDF membraneTime of incubation 1530 min at room temperature 515 min at room temperature 15 min at room temperatureSelect whenPrimary antibody is susceptibleUsing high-affinity primary antibody Using NIR-labeled antibodyto stripping buffersApplicationsDetect different targetsDesigned for use with antibodies that Gentle and highly effective reagentOptimize antibodyare difficult to remove from westernfor quickly removing primary and NIR concentrations blots, require longer incubation times, ordye-labeled secondary antibodies incubation temperatures greater than 22C from western blots70'