Stripping and Reprobing Western Blotting Membranes

Western blotting is a commonly used technique for studying protein function and localization. Typically, protein samples are separated by SDS-PAGE and transferred to a nitrocellulose or PVDF membrane which is then probed with specific antibodies. Unlike nucleic acid based technologies, which allow reuse of Southern and Northern blots, it has been difficult to reuse Western blots.

Stripping and re-probing of Western blots offers several advantages:

1. Conservation of samples that are expensive or available only in limited quantities;
2. Analysis of a given blot using several different antibodies, e.g. subtype- or isoform-specific antibodies;
3. Re-analysis of anomalous results and confirmation with the same or a different antibody;
4. Correcting errors in incubation with the wrong antibody;
5. Cost savings in reagents and time by reusing the same blot.

Several protocols for antibody stripping from Western blots have been published, including those which utilize low pH, heat and detergent, and chaotropic agents. Three recommended protocols are presented below. The first is applicable to any chemiluminescent substrate system and uses a combination of detergent and heat to release the antibodies. The second is commonly used for applications where antibodies have to be separated from an antigen and employs low pH to alter the structure of the antibody in such a way that the binding site is no longer active.

The third protocol employs the ReBlot™ Plus Western Blot Recycling Kit which has been specifically formulated for stripping antibodies from Western blot membranes. Benefits of this approach include

• Avoidance of odor related to β-mercaptoethanol.
• Room temperature processing.
• Removal of antibodies in 15 minutes.

None of these methods will remove the colored precipitates generated from chromogenic detection systems (e.g., BCIP, 4CN, DAB and TMB). However, it is still possible to analyze the blot with another antibody specific to a different target protein.

Usually, these protocols should only be used for qualitative purposes, unless it has been established that stripping does not quantitatively affect a given antigen. Depending upon the method and type of membrane used, many antigens will withstand at least 5 stripping cycles. However, it is to be taken into consideration that during each stripping cycle small portions of membrane-immobilized proteins will be removed. When several antigens are to be detected sequentially, it is recommended to start with antigens which are expected to occur at lower abundance or yield less signal.

Here are some additional recommendations when planning a Western blot experiment with one or more rounds of antibody stripping.

• PVDF membranes are more robust than nitrocellulose and are therefore recommended for any protocol involving antibody stripping.
• Drying of PVDF membranes immediately after transfer from the SDS-PAGE gel improves binding of proteins to the membrane and is particularly recommended when multiple stripping is planned. Dry PVDF membranes must be rewetted with alcohol prior to the first round of immunodetection.
• Detect low-abundance antigens first.
• Use low-affinity antibodies before high-affinity antibodies.
• Important: Although drying of a PVDF blot is recommended immediately after transfer, the blot should not be allowed to dry between rounds of immunodetection. Any residual antibody molecules will bind permanently to the membrane if it is allowed to dry.

Protocol 1 Stripping by Heat and Detergent

Required Equipment and Solutions

• Standard blot or blot strips, on nitrocellulose or PVDF membrane.
• Blocking solutions.
• Stripping solution: 100 mM 2-mercaptoethanol (M3148), 2% (w/v) SDS (L3771), 62.5 mM Tris-HCl (T6666), pH 6.7.
• Phosphate buffered saline (PBS): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaCl.
• Shallow tray, large enough to hold the membrane.
• Positive and negative stripping controls.

Procedure

1. In a fume hood, place the blot in stripping solution and incubate with agitation for 30 minutes at 50 °C.
2. Place the blot in buffer and agitate for 10 minutes. Repeat with fresh buffer.
3. Optional: Repeat the initial detection protocol (omitting the primary antibody step) to make
4. sure that the antibodies have been inactivated or stripped from the membrane.
5. Place the blot in buffer and agitate for 10 minutes.
6. Proceed to the blocking step for the next round of immunodetection.

Protocol 2 Stripping by Low pH

Required Equipment and Solutions

• Standard blot or blot strips, on nitrocellulose or PVDF membrane.
• Blocking solutions.
• Stripping solution: 25 mM glycine-HCl (G2879), pH 2,1% (w/v) SDS (L3771).
• Phosphate buffered saline (PBS): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaCl.
• Shallow tray, large enough to hold the membrane.
• Positive and negative stripping controls.

Procedure

1. Place the blot in stripping solution and agitate for 30 minutes.
2. Place the blot in buffer and agitate for 10 minutes. Repeat with fresh buffer.
3. Proceed to the blocking step for the next round of immunodetection.

Protocol 3 Stripping with the ReBlot™ Plus Western Blot Recycling Kit

Re-Blot Plus Mild Antibody Stripping Solution gives good results on both nitrocellulose and PVDF membranes. However, the Re-Blot Plus Strong Antibody Stripping Solution will perform better when membranes with high signal are to be stripped or when Re-Blot Plus Mild treatment is not sufficient.

Required Equipment and Solutions

• Standard blot or blot strips, on nitrocellulose or PVDF membrane.
• Blocking solutions.
• Plastic wrap for storage of blots that will not be reprobed immediately.
• ReBlot™ Plus Kit
• Distilled water, for reagent dilution.
• Shallow tray, large enough to hold the membrane.
• Positive and negative stripping controls.

Procedure

Note: The blots or individual strips that are to be reused should be prepared for stripping immediately after their first usage. If stripping cannot be performed right away, membranes can be wrapped in plastic wrap and stored moist in PBS at 4°C. DO NOT STORE BLOTS IN DRY FORM.

1. Fill a plastic tray with appropriate amount of 1X Antibody Stripping Solution (supplied in kit).
2. Using tweezers or forceps, submerge blot or blot strips in stripping solution. Incubate with gentle mixing for 15 minutes at room temperature.
3. Fill a clean plastic tray with an equal amount of blocking buffer. Conventional blocking buffers such as 20 mM Tris HCl, pH 8.0; 150 mM NaCl; 0.1% TweenR-20; 5% dry milk or similar are appropriate.
4. Wash blots two times 5 minutes each with blocking buffer.
5. The blot is now ready for reprobing with antibodies.

References

1. Lioubin MN, Myles GM, Carlberg K, Bowtell D, Rohrschneider LR. 1994. Shc, Grb2, Sos1, and a 150-kilodalton tyrosine-phosphorylated protein form complexes with Fms in hematopoietic cells.. Mol. Cell. Biol.. 14(9):5682-5691. https://doi.org/10.1128/mcb.14.9.5682

2. Legocki RP, Verma DPS. 1981. Multiple immunoreplica technique: Screening for specific proteins with a series of different antibodies using one polyacrylamide gel. Analytical Biochemistry. 111(2):385-392. https://doi.org/10.1016/0003-2697(81)90577-7

3. Harlow E, Lane D. 1988. A Laboratory Manual. 579. New York: Cold Spring Harbor Laboratory.

4. Kaufmann SH, Ewing CM, Shaper JH. 1987. The erasable Western blot. Analytical Biochemistry. 161(1):89-95. https://doi.org/10.1016/0003-2697(87)90656-7

5. Yeung Y, Stanley ER. 2009. A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing. Analytical Biochemistry. 389(1):89-91. https://doi.org/10.1016/j.ab.2009.03.017