Thermo Scientific™ Restore™ PLUS Western Blot Stripping Buffer

After stripping with Restore Buffer, re-blocking of the membrane is usually not necessary but may help to decrease background in some situations. By contrast, re-blocking is required after stripping with Restore Plus Buffer.

Yes. The stripping buffers work to separate the antibody from the antigen, so the membrane to which the antigen is bound generally will not affect the stripping.

Yes. Both incubation time and temperature must be optimized for best results.

No. Every binding interaction is slightly different in terms of mode (i.e., actual amino acids or functional groups that interact) and affinity (strength of binding under a given set of buffer conditions). First try Restore Western Blot Stripping Buffer for 15 minutes at room temperature. If antibody removal is incomplete, optimize the stripping conditions by increasing the time and temperature. If this fails to completely strip the antibodies, then switch to the more stringent Restore Plus Western Blot Stripping Buffer.

Restore Western Blot Stripping Buffer gently but effectively removes the primary and secondary antibodies from the membrane to allow re-probing on the same membrane. The buffer is formulated to work for a wide variety of interactions, but there are some high affinity antigen-antibody interactions that require more stringent stripping conditions. Restore Plus Western Blot Stripping Buffer was developed for these difficult to strip interactions.

There are two types of stripping buffers, each available in two different sizes:

• Restore Western Blot Stripping Buffer: Cat. No. 21059 (500 mL), Cat. No. 21063 (5 L) and Cat. No. 21062 (30 mL)
• Restore Plus Western Blot Stripping Buffer: Cat. No. 46430 (500 mL) and Cat. No. 46428 (30 mL)

For nitrocellulose or PVDF membrane following Western blot detection using a chemiluminescent or fluorescent substrate system: Following transfer, air dry the membrane and place in an envelope, preferably on top of a supported surface to keep the membrane flat. The blot can be stored indefinitely at -80 degrees C. When ready to reprobe, prewet the PVDF blot with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with blocking step.

FOR STRIPPING/REPROBING OF MEMBRANES: Harsh protocol (see NOTE below for modifications)

1) Submerge the membrane in stripping buffer (100 mM BME, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubate at 50 degrees C for 30 min with occasional agitation. If more stringent conditions necessary, incubate at 70 degrees C.

2) Wash 2 x 10 min in TBS-T/PBS-T at room temperature.

3) Block the membrane by immersing in 5% blocking reagent TBS-T or PBS-T for 1 hr at room temperature.

4) Immunodetection

NOTE: Often you don't need such harsh conditions to remove antibodies from their proteins. The stringency of one or several of the variables can be decreased: lower the temperature, decrease the time, less BME, less SDS, etc. An especially mild but still often effective stripping protocol is lower pH incubation. Example: pH 2.0 Tris 50-100 mM, 30-60 min incubation (you may do two incubations if you wish). Then rinse and block as usual. If you do not wish to re-use the membrane immediately after stripping, you can store the membrane in plastic wrap (wet, you do not want it to dry out). Another simple, mild stripping buffer is 0.1 M glycine•HCl (pH 2.5-3.0), incubation 30 min to 2 hrs room temperature or 37 degrees C, depending on the antibody.

Western blots can be stripped by incubation in Restore Western Blot Stripping Buffer at 37 degrees C for 5-15 min. If more stringent conditions are required, Restore Plus Western Blot Stripping Buffer can be used. Alternatively, membranes can be incubated in a stripping buffer consisting of 100 mM BME, 2% SDS, 62.5 mM Tris-HCL, pH 6.7, at 50 degrees C for 30 min with agitation. Wash the blot 3 times for 10 min each in PBST at room temperature. To reprobe with your antibody, the blot will need to be blocked again for 1 hr at room temperature.

Another stripping buffer is 0.1 M glycine-HCl (pH 2.5-3.0), the same solution commonly used for elution in immunoaffinity protocols. This solution will dissociate most antibody:antigen interactions at room temperature or 37 degrees C, but for strong antibody:antigen recognition, a 2 hr incubation may be necessary.