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查看更多产品信息 Restore™ Western Blot Stripping Buffer - FAQs (21059, 21059X4, 21063, 21062)
15 个常见问题解答
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Wash the membrane with the same buffer as was used between antibody probing steps during the Western blotting procedure.
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No. Although the fluorescent antibodies, like other antibodies, can be stripped with Restore Buffers, stripped membranes typically produce unacceptable background for subsequent fluorescent detection methods. We recommend Restore Fluorescent Western Blot Stripping Buffer, Cat. No. 62299 (20 mL) and Cat. No. 62300 (100 mL).
Note: Restore Fluorescent Western Blot Stripping Buffer is for use with low-fluorescence PVDF membrane (Cat. No. 22860) only.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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. However it may be possible to strip the complexed biotinylated primary antibody and streptavidin-HRP secondary antibody from the target protein on the membrane. Please also see Tech Tip: Strip and Reprobe Western Blots (https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/strip-reprobe-western-blots-tech-tip.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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. Please note that this is not compatible with fluorescence supstrates as it will result in increased background. For fluorescent substrates please use our Restore Fluorescent Western Blot Stripping Buffer. Please also see Tech Tip: Strip and Reprobe Western Blots (https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/strip-reprobe-western-blots-tech-tip.pdf).
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
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Yes. Both incubation time and temperature must be optimized for best results.
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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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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)
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Nitrocellulose and PVDF membranes can be stripped following detection with SuperSignal chemiluminescent substrates, for reprobing. Restore Western Blot Stripping Buffer (Cat. No. 21059, 21063) can be used to gently but efficiently remove probes from the western blot membrane without damaging the target protein. In most cases, re-blocking of the membrane is not necessary.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Nitrocellulose and PVDF membranes can be stripped following detection with SuperSignal chemiluminescent substrates, for reprobing. Restore Western Blot Stripping Buffer (Cat. No. 21059, 21063) can be used to gently but efficiently remove probes from the western blot membrane without damaging the target protein. In most cases, re-blocking of the membrane is not necessary.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.