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View additional product information for Restore™ Fluorescent Western Blot Stripping Buffer - FAQs (62299, 62300, 62300X4)
10 product FAQs found
粘附（转印和结合的）蛋白的稳定性决定了膜在剥离后可成功再次检测的次数。蛋白质可承受剥离的次数最多4次、最少1次。
洗膜缓冲液应与蛋白质免疫印迹中抗体检测步骤之间所用的缓冲液相同。
不能。尽管荧光抗体，如其他抗体，可被Restore缓冲液剥离，但被剥离的膜在后续荧光检测中通常会产生较高的背景。我们建议使用Restore Fluorescent Western Blot剥离缓冲液，货号62299（20 mL）和货号62300（100 mL）。
注意：Restore Fluorescent Western Blot剥离缓冲液仅能用于低荧光PVDF膜（货号22860）。
不能。抗体可以被去除，但底物会永久沉淀在膜上，不能被去除。Restore和Restore Plus缓冲液专为利用化学发光底物的实验过程而设计。
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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. 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.
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.