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Western Blot Stripping Blots

Western Blot Stripping Blots - Western Blot Forum

Western Blot Stripping Blots - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 01-28-2009, 04:32 AM
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Question Western Blot Stripping Blots



Hi, ive a blot that I'll work on first with Primary Ab that is monoclonal raised from goat. Then as secondary Ab, I'll use "rabbit anti-goat Ab".

Next I am planning to work on the same blot, this time with Primary Ab that is monoclonal raised from rabbit. Then secondary Ab, I'll use "Goat anti-rabbit Ab".

Do you think I should strip blot for the latter before proceeding? What worries me is that the secondary "Goat anti-rabbit" will interact with residual of secondary "Rabbit anti-goat".

Also do you think the protein interaction of secondary to primary Ab will break during the re-probing steps or that it's better to just STRIP?
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Old 01-28-2009, 04:32 AM
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Default Re: Western Blot Stripping Blots

Hi again, I would definitely strip the western blot membrane before re-probing with another antibody
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Old 01-28-2009, 04:33 AM
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Default Re: Western Blot Stripping Blots

Hi, you may succeed without stripping but you should inactivate rest activity of peroxidae (if you use HRP-labeled 2nd Ab) of first round by incubation with NaN3; then wash extensively. However I think your goat anti-rabbit will bind to the rabbit anti-goat... im getting confused...
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Old 03-31-2011, 07:54 PM
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Default Re: Western Blot Stripping Blots

if you wait for a day or two in between your two probes it should not give you any trouble.I don't think these two 2nd Abs would cross-react,but who knows we always get unexpected backounds.You can go for two consequtive probings after a decent interval if you use two 2nd Abs of separate origins.But my suggestion is stripping is the better option.Good luck.
MB
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