Signal-to-noise ratio (S/N ratio) refers to how much relevant content (signal) something has as opposed to non-relevant content (noise). In Western blotting, the signal is the density of the specific protein band being probed for; the noise is the density of the background. Optimizing the S/N ratio is often more important than increasing the sensitivity of the system. The sensitivity of the system is irrelevant if the signal cannot be distinguished from the noise. When required, optimization of a Western blot assay is time consuming and can exhaust valuable samples. Reprobing a Western blot save times and conserves sample while allowing optimization to be performed as needed. Reprobing also allows the same blot to be probed for different target proteins.

Watch this video on preparing blots to reprobe


One of the major advantages offered by chemiluminescent detection is the ability to strip reagents from a blot and then reprobe the same blot. With chemiluminescence, all of the reagents can be removed from the membrane since the product detected is light rather than a colored precipitate or a fluorescent molecule absorbed on the membrane. A blot may be stripped and reprobed several times to visualize other proteins or to optimize detection of a protein (i.e., antibody concentrations) without the need for multiple gels and transfers.

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Advantages of Reprobing Blots

Major reasons researchers choose to strip and reprobe Western blots
Conserves sample When the protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be analyzed 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 optimization is easier The light emission intensity of high sensitivity chemiluminescent substrates often require antibody concentration optimization to achieve the highest quality blot. Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration.
Quickly confirm atypical results When immunoblot results are not as expected, reprobing allows the use of the same protein sample without going back to gel electrophoresis.
Correct mistakes Immunoblotting requires many steps, providing ample opportunity for mistakes to occur. By stripping the membrane, the blot can be reused.

Stripping Western Blot Membranes

The key to stripping a membrane is to use conditions that cause the release of antibody from the antigen without causing a significant amount of antigen to be released from the membrane. Various protocols have been developed to accomplish this purpose and they generally include some combination of detergent, reducing agent, heat and/or low pH. During the stripping procedure, some amount of antigen is inevitably lost from the membrane. It is important to minimize this loss by stripping the antibody under gentle conditions. Because each antibody-antigen pair has unique characteristics, there is no guaranteed method to remove every antibody while preserving the antigen.

Reprobing with different antibodies. Western blots of HeLa cell lysate protein (diluted from 750ng down to 83.3ng) were detected with SuperSignal West Dura Chemiluminescent Substrate. The first blot used polyclonal rabbit anti-JAK-1 primary antibody (BD PharMingen; San Jose, CA) at 1:2000 dilution with an HRP-secondary conjugate diluted at 1:350,000. The same blot was stripped for 5 minutes at room temperature in Restore Western Blot Stripping Buffer and then reprobed with purified Mouse Anti-Human Bak monoclonal primary antibody at 1:1000 with the HRP-secondary conjugate at 1:100,000. Five percent nonfat milk with 0.05% Tween 20 was used for blocking.

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Testing and Reprobing Stripped Blots

Following any stripping procedure, the blot should be tested to ensure that all of the detection reagents were removed. The membrane should be washed several times, blocked, incubated with secondary antibody and then reincubated with chemiluminescent substrate. If the primary antibody was effectively removed by the stripping procedure, no secondary antibody should bind to the membrane and no signal should be produced. If bands are still visible on the blot, the stripping conditions must be intensified. Often a simple increase of the reaction time or temperature will complete the stripping process. However, it is sometimes necessary to alter the composition of the stripping buffer or change methods entirely.

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  • Tech Tip #24: Optimize antigen and antibody concentrations for Western blots

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