WB antibody to be used in IHC - (May/27/2008 )

Hi!

I was wondering.. could I use the antibody specified for WB for IHC? The product datasheet only mentioned to be used for WB, but I don't have much choice for the antibody. . I scared that I could not get result..

What should I do?

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You could just give it a shot - what have you got to lose? Perhaps the data sheet says WB because they haven't tested it for IHC? Or they have and it doesn't work. Just make sure the antibody does not have azide in it - this will inhibit the HRP (if you use a HRP secondary antibody).

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Correct me if I am wrong, but I don't think azide matters. There will be extensive washing between primary and secondary.

And yes, all antibodies are not tested for all applications, unless they specifically mention that it does not work for IHC, you volunteer

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[/quote]
Correct me if I am wrong, but I don't think azide matters. There will be extensive washing between primary and secondary.

And yes, all antibodies are not tested for all applications, unless they specifically mention that it does not work for IHC, you volunteer
[/quote]

I think it does (in my hands anyway). I made a monoclonal antibody and added azide to keep it happy for long-term storage in 4deg. My IHC did not work So then someone told me that azide inhibits the reaction, so I dialysed the azide out and wOOt! my IHC worked I can't remember what concentration of azide I used, so perhaps really low concentrations don't matter?

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I feel for ya oops..wrong smiley.

No, I don't really know, I am just hypothesizing; perhaps even the homeopath dose of azide inhibits reaction for all that I know.

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Thank you for all your reply

Well I guess I don't have much choice ... though the antibody is kinda expensive..
So is it fine to use other application even not mentioned in the product datasheet?
So far, I keep play it safe by checking the product info.

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Best thing is to look for literature and references if somebody has used it for the purpose. Otherwise, just dive..

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western blot is usually with denatured protein, ihc is with native protein.

you can try a dot blot with native protein before wasting the sample.

if the antibody is polyclonal then there is a greater chance that the antibody will work with native protein.

if it is monoclonal then you have to hope (or know) that the epitope is exposed in the native protein.

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