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 saves time and conserves sample while allowing optimization to be performed as needed. Reprobing also allows the same blot to be probed for different target proteins.

Introduction

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.

Watch this video on preparing blots to reprobe


Western blot stripping procedure. HeLa tumor cell lysates were probed with actin antibodies and detected with Thermo Scientific Pierce ECL Western Blotting Substrate. Blots were then stripped with Thermo Scientific Restore PLUS Western Blot Stripping Buffer. The blot was then re-blocked and reprobed with cyclochilin B antibody and detected with Pierce ECL substrate.


Protein Detection Technical Handbook

This 84-page handbook provides a deep dive into the last step in the western blot workflow—protein detection. With a variety of detection techniques to choose from (chemiluminescence, fluorescence or chromogenic), you can select a technology to match your experimental requirements and the instruments you have available. Whether for quick visualization or precise quantitation, single-probe detection or multiplexing—Thermo Fisher Scientific offers a range of reagents and kits for western blot detection and subsequent analysis.

Request download

Learn more


Advantages of reprobing blots
There are several major reasons researchers choose to strip and reprobe a western blot. Following are some of the most important:
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 750 ng to 83.3 ng) were detected with Thermo Scientific SuperSignal West Dura Extended Duration Substrate. For the first blot, JAK-1 was detected with a rabbit anti-human JAK-1 polyclonal antibody at 1:2,000 dilution followed by detection with a horseradish peroxidase (HRP)-secondary conjugate diluted at 1:350,000. The same blot was stripped for 5 minutes at room temperature in Thermo Scientific Restore Western Blot Stripping Buffer. To detect BAK protein, the blot was reprobed with mouse anti-human BAK monoclonal antibody at 1:1,000 followed by detection with HRP-secondary conjugate at 1:100,000. Five percent nonfat milk with 0.05% Thermo Scientific Tween 20 detergent solution was used for blocking.


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 re-incubated 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.

A. Western blot analysis

B. Densitometry

Western blot stripping and reprobing. Comparison of stripping buffers on nitrocellulose. Three different concentrations of HeLa cell lysate were separated by SDS-PAGE and transferred to a to 0.45-μm nitrocellulose membrane. (A) After blocking, the membrane was analyzed by western blot using SuperSignal West Dura Extended Duration Substrate. The membrane was probed with anti-Hsp90 polyclonal antibody followed by goat anti-rabbit horseradish peroxidase conjugate and imaged. Following the initial detection, the blot was cut into three sections, and stripped, according to the manufacturer’s instructions, in either Restore Western Blot Stripping Buffer, Reblot Plus Stripping Solution, or Revitablot Western Blot Stripping Buffer. Following the stripping procedure, the membrane sections were washed, incubated with substrate, and imaged. The membrane sections were reblocked and the western blot procedure repeated as described above. (B) Densitometry analysis shows that the Restore stripping buffer permitted both complete signal removal and maintained nearly identical levels of detection between the initial and reprobed western blot analysis.


Recommended reading

Alegria-Schaffer A, Lodge A, Vattem K. (2009) Chapter 33: Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection. Methods in Enzymology 463:573–599.