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View additional product information for Restore™ PLUS Western Blot Stripping Buffer - FAQs (46430X4, 46430, 46428)
24 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。Restore和Restore Plus缓冲液的剥离力度不足以破坏抗生物素蛋白-生物素相互作用。能够破坏这一相互作用的缓冲液也可能会损害目标蛋白，使其无法被检测到。
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。
使用Restore缓冲液剥离后的膜，通常无需再次封闭，但是再次封闭在某些情况下可能有助于降低背景。相反，使用Restore Plus缓冲液剥离后的膜需要再次封闭。
可以。剥离缓冲液是用于从抗原上分离抗体的，因此，结合抗原的膜通常不会影响剥离。
需要。为获得最佳结果，孵育时间和温度都必须优化。
未进行测试。每种结合相互作用的模式（即，实际发生相互作用的氨基酸或功能性基团）和亲和性（在给定缓冲液条件下的结合强度）都稍有不同。首先，尝试使用Restore Western Blot剥离缓冲液在室温下剥离15分钟。如果未完全去除抗体，则通过延长时间和升高温度对剥离条件进行优化。如果仍未完全剥离抗体，则改为使用更强力的Restore Plus Western Blot剥离缓冲液。
Restore Western Blot剥离缓冲液可温和而有效地从膜上剥离一抗和二抗，从而对同一张膜进行再次检测。该缓冲液配方可有效用于多种相互作用，但是，有些高亲和性抗原-抗体相互作用需要更强力的剥离条件。Restore Plus Western Blot剥离缓冲液专为难剥离的相互作用而开发。
有两种剥离缓冲液，每种都具有2种不同规格：
•Restore Western Blot剥离剥离缓冲液：货号21059（500 mL），货号21063（5 L）和货号21062（30 mL）
•Restore Plus Western Blot剥离剥离缓冲液：货号46430（500 mL），货号46428（30 mL）
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. 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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes. Both incubation time and temperature must be optimized for best results.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.