Stripping Blots Protocols

[moleculardude]

Hello everyone.

I have some western blot stripping protocols to share.



To Strip Blots, make stripping buffer.

For 50 mL:

Add...

100 mM 2-mercaptoethanol 0.35 ml
2% SDS 10 ml of 10% SDS
62.5 mM Tris, pH 6.8 12.5 ml of 0.25 M Tris, pH 6.8

Shake blot gently at 50C for 30 min
Wash 2X with TBS-Tween

Re-block in milk for at least 1 hour after stripping
Re-probe blot with primary and secondary antibodies

[kliron]

A less harsh (and stench-free :-)), but VERY efficient alternative:
wash blot for 5 min in distilled water
immerse in 0.2M NaOH and wash for 5 minutes
wash again for 5 minutes in distilled water
Block for 30-60 min
If any trace of signal still remains you can raise the concentration of NaOH up to 2M and the wash time up to 30 min (although I 've never needed to...).

[fitbrit]

Thanks for these- we may well be needing to do this soon. I'll try the NaOH method first.

[bwbrian]

0.2 M NaOH isn't really very gentle either. I'm busy (lazy?), so I buy Restore stripping buffer from Pierce. It doesn't have the odor of mercaptoethanol and I haven't had problems with losing signal because it's too harsh.