i am using stripping buffer for 1hr to remove the previous bands but still the previous band are visible. is it possible to completely remove the previous bands with stripping buffer ? .
Yes, it's possible !
Try to use this protocol:
1) After the previous development of the membrane with your Ab and ECL reagents, incubate the membrane with water 3 times by 5 min on the rotating platform.
2) Wash the membrane in the buffer containing 0.1 M Glycine-HCl, 0.5 M NaCl, pH 2.7 for a few seconds.
3) Remove your buffer and incubate your membrane in the same buffer containing 0.1 M Glycine-HCl, 0.5 M NaCl, pH 2.7 for 10 min on the rotating platform.
4) Incubate the membrane after that again in water 3 times by 5 min changing water each time.
5) Incubate the membrane in the same buffer where your next 1st Ab is going to be dissolved for a few seconds.
6) Incubate your membrane in the buffer where your next 1st Ab is dissolved and go on further with your procedure.
Hope it will be helpful.
Dear Imdad
I agree with Victors prior answer and this probably represents the best approach:
1. Aggressive stripping with buffers for prolonged time periods like 1 hour (as you mentioned) but pre heating the buffer to 60C can more effectively strip bands in order to re probe than prolonged incubation @ room temp alone but in my experience can also strip some of your bound lysate protein
2. I would therefore just add that when I western blot; strip and re probe I tend to identify GOI with primary antibody; strip and then re probe with a housekeeping antibody like B tubulin or B actin (to confirm data normalisation) second; Done the other way would require more aggressive stripping of very strong bands and that can distort re probing for much weaker bands. The same principle applies to detection of anticipated weak and strong GOI signals. Do the former first
3. Also in my experience it is better just to strip and re probe once and not successively strip and probe for a plethora of genes as this can definitely lead to denuding of protein from the membrane and in general increased back ground problems
It solely depends on the antibody. Some antibodies are extremely strong (especially if the protein is abundant) and never goes away completely, other are weak and the membrane comes back clean.
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