Currently I am using from Thermo Scientific, however, it is unable to strip mouse Abs and very low potency in rabbit Abs. Do anyone have good experiences with commercially available stripping buffer for fluorescent western? Your suggestions would really be appreciated.
I use LiCor's NewBlot NitroStripper.
https://www.licor.com/bio/products/reagents/stripping_buffers/newblot_nitro.html
It's pretty easy to tweak it beyond just diluting (using Type 1 water, it'll be at full-strength at a given dilution, but if you use a buffer system, it weakens considerably).
I will say it's more finicky than HRP stripping buffers. Some of what I probe for comes off in five minutes and no matter what intensity I use, I can't see it. Other proteins simply do not strip off unless I use conditions that may as well have me toss the blot.
Hi Moniruzzaman,
Try this out.
" To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H20. Make buffer fresh just prior to use. "
Good Luck..!
https://www.cellsignal.com/contents/resources-protocols/western-blot-reprobing-protocol/western-reprobing
We generally use 0.1 M NaOH to strip blots. (20-30 min with gently shaking). However, we also have one or two primaries that are difficult to completely remove. One solution could be to probe for the target that is 'strippable' first. Another approach, if your imaging system permits, would be to get an anti-mouse secondary that is conjugated with a different fluorophore, with distinct excitation and emission wavelengths, and use it if you need to reprove with a different mouse antibody.
We observed that placing the PVDF membrane in an incubator with agitation at 55ºC for 1 h with the Thermo Restore buffer helped. But we found that some sticky antibodies resisted.
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