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.
Stripping and reprobing a western blot is a method in which the primary and secondary antibodies are removed from a western blot so the blot can be reprobed. 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.
Explore: Stripping Buffers Stripping and reprobing protocols
Why strip and reprobe?
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.
Advantages of reprobing blots
There are several major reasons researchers choose to strip and reprobe a western blot. The 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 the gel, membrane, and protein sample. |
| Easier assay optimization | 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. |
Western blot stripping example. 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 cyclophilin B antibody and detected with Pierce ECL substrate.
How to strip and reprobe 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 (protein) 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.
Things to consider before getting started with stripping membranes to be reprobed:
| Use PVDF membranes | PVDF membranes have a protein binding capacity of 170–200 µg/cm2 and offer better retention of adsorbed proteins than other supports because of the greater hydrophobicity. PVDF is less brittle and fragile than nitrocellulose, making it more useful when requiring multiple rounds of reprocessing (stripping and reprobing procedures). |
| Strip and reprobe only chemiluminescent and fluorescent western blots | Colorimetric/chromogenic detection reagents leave a permanent visible stain on the membrane that will not be removed by stripping procedures. |
| Start with mild conditions | Start with gentle, mild conditions to avoid loss of sample. Use more stringent conditions if required to break up strong antigen-antibody pairs. |
| Probe for low-abundance proteins first | With every round of stripping, there is a potential for antigen loss. Minimize these effects by probing for low-abundance proteins or with low-affinity antibodies first. |
| Avoid avidin-conjugated probes | The bond between streptavidin and biotin is very strong and can be difficult to dissociate for reprobing. |
Mild stripping of western blot membranes
Mild stripping buffer recipe
| Glycine | 15 g |
| SDS | 1 g |
| Tween 20 | 10 mL |
| Deionized water | 800 mL |
| pH to 2.2 with HCl | |
| Deionized water | to 1000 mL |
 | Ready to use alternative:Restore Western Blot Stripping Buffer or Restore Fluorescent Western Blot Stripping Buffer |
Mild stripping protocol (Stripping by low pH)
- Rinse membrane in water to remove excess chemiluminescent substrate on the membrane.
- Incubate the membrane protein-side up in the stripping buffer with agitation, for 10-20 minutes at room temperature. Ensure the volume of the stripping buffer is enough to fully cover the membrane.
Optimization of both incubation time and temperature is essential for best results. For some antibodies, room temperature incubation is sufficient. However, high-affinity antibodies or saturated blots (excess secondary antibody) may require incubation for an additional 5 to 10 minutes at 37°C.
- Wash the membrane 3 times with agitation for 5 minutes each in wash buffer (TBST or PBST).
- Recommended: proceed to reblocking the membrane prior to reprobing.
Stringent stripping of western blot membranes
Stringent stripping buffer recipe
| 0.5 M Tris HCl, pH 6.8 | 12.5 mL |
| 10% SDS | 20 mL |
| 2-mercaptoethanol | 0.8 mL |
| Deionized water | 67.5 mL |
| Make buffer just prior to use in a fume hood | |
| Ready to use alternative:Restore PLUS Western Blot stripping buffer |
Stringent Stripping Protocol (stripping by heat and detergent)
- Rinse membrane in water to remove excess chemiluminescent substrate on the membrane.
- Incubate the membrane protein-side up in the stripping buffer with gentle agitation, for 30 minutes at 50 °C in a fume hood. Ensure the volume of the stripping buffer is enough to fully cover the membrane.
- Wash the membrane 6 times with agitation for 5 minutes each in wash buffer (TBST).
- Proceed to reblocking the membrane prior to reprobing.
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.
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.
Additional Resources
For Research Use Only. Not for use in diagnostic procedures.