Stripping membrane of Western blots - Introduction (Oct/31/2006 )
i'm looking for info about stripping membranes after WB...all what i found are methods and protocols....that's fine, but i'm looking for a good introduction on this topic since it's new to me..
any help would be appreciated...
Use "Restore" from Pierce Scientific. Works very well. They have all the technical info on their website.
How do you use it because I have not been able to get it to work for me.
Could you not get it to work because you could not strip the membrane or because you could not detect protein when you reprobed with a different antisdy. These are signs that either you were in the buffer too short or too long, respectively. Generally, let the membrane sit in the stripping buffer about 15-20 minutes and then wash with pbs. You then need to reblock and then reporbe with antibody. You may have to work out the conditions for different antibodies. See the instructions, they are detailed.
Maybe my antibody was just happy where it was because I got some of it to come off, but I could still see bands from my previous run alongside new bands from my new antibody. I will try incubating it longer next time...
thanx tap14, i'll check it..
any good introductory sites?
You can also increase the stripping by doing it at 37 degrees.
Here is a link for the buffer from the Pierce Website. You need acrobat reader to open.
This is a good website from Abcam, it contains some helpful protocols for WB.
this is from pierce site
Stripping and Reprobing a Membrane
One of the major advantages offered chemiluminescent detection offers is the ability to strip reagents from a blot and then reprobe the same blot. With chemiluminescence, all of the reagents can be removed from the membrane since the product detected is light rather than a colored precipitate on the membrane. A blot may be stripped and reprobed several times to visualize other proteins or to optimize detection of a protein (i.e., antibody concentrations) without the need for multiple gels and transfers. The key to this process is to use conditions that cause the release of antibody from the antigen without causing a significant amount of antigen to be released from the membrane. Various protocols have been proposed to accomplish this purpose and they generally include some combination of detergent, reducing agent, heat and/or low pH. During the stripping procedure, some amount of antigen is inevitably lost from the membrane. It is important to minimize this loss by stripping the antibody under gentle conditions. Because each antibody-antigen pair has unique characteristics, there is no guaranteed method to remove every antibody while preserving the antigen. Restore Western Blot Stripping Buffer (Product # 21059) was designed to achieve maximum removal of antibodies from a membrane while preserving the integrity of the antigen. It is unique among stripping buffers because it is odor-free and can often strip a membrane in as little as 15 minutes.
Following any stripping procedure, the blot should be tested to ensure that all of the detection reagents were removed. The membrane should be washed several times with blocking agent, incubated with secondary antibody, then reincubated with chemiluminescent substrate. If the primary antibody was effectively removed by the stripping procedure, no secondary antibody should bind to the membrane and no signal should be produced. If bands are still visible on the blot, the stripping conditions must be intensified. Often a simple increase of the reaction time or temperature will complete the stripping process. However, it is sometimes necessary to alter the composition of the stripping buffer or change methods entirely.
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