What stripping buffers are available?
There are two types of stripping buffers, each available in two different sizes:
Restore Western Blot Stripping Buffer: #21059 (500 ml) and #21062 (30 ml)
Restore Plus Western Blot Stripping Buffer: #46430 (500 ml) and #46428 (30 ml)
What is the difference between the Restore and Restore Plus Stripping Buffers?
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.
Have the stripping buffers been tested for my primary or secondary antibody?
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.
Do I need to re-optimize conditions when switching from Restore to Restore Plus Buffer?
Yes. Both incubation time and temperature must be optimized for best results.
Can Restore and Restore Plus Buffers be used on both nitrocellulose and PVDF membranes?
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.
Do I need to re-block the membrane after stripping?
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.
Will the stripping buffers remove precipitating substrates?
No. The antibodies are removed but the substrate leaves a permanent precipitate on the membrane that cannot be removed. Restore and Restore Plus Buffers are designed for procedures using chemiluminescent substrates.
Will stripping buffers remove biotin-bound avidin, streptavidin or NeutrAvidin Protein conjugates?
No. Restore and Restore Plus Buffers are not stringent enough to break the avidin-biotin interaction. A buffer capable of breaking this interaction would likely also damage the target protein, making it undetectable.
Can I strip and reprobe membranes for subsequent detection with fluorescent probes?
No. Although the fluorescent antibodies, like other antibodies, can be stripped with Restore Buffers, stripped membrane typically produce unacceptable background for subsequent fluorescent detection methods.
What is the proper wash buffer to use after stripping?
Wash the membrane with the same buffer as was used between antibody probing steps during the Western blotting procedure.
How many times can I strip my blot?
The stability of the attached (transferred and bound) protein will determine the number of times the membrane can be successfully re-probed after stripping. The protein may withstand stripping as many as four times or as few as one time.
Can Restore Buffers be used in the Pierce In-Gel Chemiluminescent Detection System?
Yes. Restore Buffer can be used for gels, following the same protocol described for membranes. However, Restore Plus Buffer has not been tested with the Pierce In-Gel Chemiluminescent Detection System.