Stripping for Western blots - What is the common procedure for stripping? (Apr/17/2008 )

Dear all,

1) I need some help or opinion in western blot or stripping. I had stripped my blots and left them in 4C in blocking buffer. How long can I keep the blots?

2) And how many times can I strip the blots until I cant use it anymore? My protein of interest is about the similar size. Thus I am not sure if I can strip them more than twice.

3) Which stripping buffer is recommended? Apparently there are two types of stripping buffer and some uses mercaptho and some not. WHich is better? I know the one with mercaptho has stronger effect of stripping the blots.

Any input is much appreciated.

Thanks

I haven't stripped a membrane so far so i don't have experience.In our lab protocol the stripping solution is the one with mercaptethanol.
Maybe a quick test with ponsius dye can help you check the effect of stripping.
As for the storage personally i use a sealed container with TBS-tween 20 1%@ 4C.I think that blocking solution can be easily contaminated.
I hope i helped you in a way

Stripping is can be very tough on a membrane and remove sample proteins, hence loosing your signal and making further blotting difficult or impossible. I only strip blots when absolutely necessary and I never tell my boss that a blot was stripped (he thinks it's evil and can completely change a membrane, therefore unbelieving any data from a stripped blot).

If you can use antibodies from different species, you can always use sodium azide to kill the HRP from the first probe and then just reprobe with a different species. Otherwise, I recommend the commercial from Pierce. It seems to be the most effective while not damaging the membrane too badly. However, some antibodies bind so well that they won't strip without the more harsh method with mercaptho.

Leaving a membrane in blocking too long isn't good. Bacteria can grow and destroy the blot. For long term storage, dry the membrane completely and store in plastic-wrap. You can always rehydrate and reprobe later. I've taken blots from 2 years ago and had them still work. Otherwise, store in buffer (TBST or PBST) and be sure to change the buffer every day or two to avoid bacterial growth or add a bit of sodium azide (or even antibiotics) to stop growth.

As for how many times you can strip, depends on how strong the stripping was, how abundant the protein was in the lysate, how sensitive the antibody is, ect. Every time you strip you take a bit of the proteins from the sample away making it more and more difficult. Start with the least sensitive antibody and work your way to the most sensitive. I've seen some membranes strip over 5 times and still give signal. Others wouldn't give a signal after one strip.

As a general rule, I think it's best to start at the least harsh method, check for residual signal and continue stripping if needed. It's easy to continue stripping or up the method to a more stringent strip if needed but once you take away the sample or destroy the membrane, it's trash.

I have done the mercaptoethanol stripping a couple of times, and it works OK out to about 3-4 times, beyond that I don't really trust the results much. I have seen it go quite wrong as well, if you are not careful some parts of the membrane can be stripped more than others, so I am always a bit dubious about stripped membranes.