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


western blot validation

western blot validation - Western Blot Forum

western blot validation - 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 06-18-2011, 03:38 PM
Pipette Filler
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Default western blot validation



I am attempting to show the ELISA data we have is actually measuring the antigen we say it is. Since most of the time we don't have a species specific kit for example human kit to measure canine serum.
So I've attempted to strip the ELISA wells after being incubated with serum and running the antigen on a western blot. I found out quick that didn't work. So now I'm trying to use the antibody from the ELISA kit on a western blot to show specificity. The down side I can only use this method on sandwitch ELISA's. I need to do the same for a competitive ELISA. Ideally I would just mas spec the protein, but the mass spec is being worked on and they want the results sooner rather than later.
Is my thought process correct would antibody specificity be enough? Any ideas on another way to go about this?
Any help would be greatly appreciated.
Thanks
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Old 08-08-2012, 02:21 PM
Pipette Filler
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Default Re: western blot validation

Nevermind. The idea was a failure from the start. There is not near enough protein captured in an elisa well, to be able to strip, dialyze, concentrate, and be able to run on a western blot.
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