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same primary


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#1 Ger

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Posted 29 September 2004 - 01:29 PM

Hi there
I am interested in determining 2 proteins interact.
Unfortunately the primary antibodies for both proteins come from the same species.
Is there any way I can do immunostaining or Immunoprecipitation now?
Is IP OK or will that mess it up?

Thanks a lot!

#2 jadefalcon

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Posted 30 September 2004 - 06:16 AM

when you primary abs are from the same species, you will not be able by dual immunostaining to differentiate which protein is which.
so I would recommend two seperate immunostains from to consecutive cuts of your material, one with the 1st prime-ab, the other withe the 2nd prime-ab. since two consecutive layers are quite similar, you should be able to get two pictures theat should be easy to overlap.

if your proteins are diferent in size , i.e. seperable be PAGE, IP should give you no headaches. Co-IP for the identification of interaction partners will not work, though, as you have no way of telling where the interaction came from. so either you do two seperate samples as above, or you IP your two proteins together, depends on what you're up to.

mike
--- He who finds typos may keep them! ---

#3 Sprag

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Posted 04 November 2004 - 08:45 AM

It is possible to detect co-localisation by immunofluorescent. You simply stain up individually with primary then secondary (FITC), then incubate with the other primary, and then use a second secondary (TRITC)... I've tried that many times without any problems...

Also by IP... unfortunately you can't avoid the two huge Ig bands at 50 and 25 kDa!! Unless you use a special secondary antibody called TrueBlot.. these antibodies do not recognise reduced IgG...

good luck,

#4 yang5yc

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Posted 21 January 2005 - 01:02 PM

It is possible to detect co-localisation by immunofluorescent. You simply stain up individually with primary then secondary (FITC), then incubate with the other primary, and then use a second secondary (TRITC)... I've tried that many times without any problems...

Also by IP... unfortunately you can't avoid the two huge Ig bands at 50 and 25 kDa!! Unless you use a special secondary antibody called TrueBlot.. these antibodies do not recognise reduced IgG...

good luck,

Hi Spraq,

I'm glad to hear that you are doing double staining and with no problem with it. My question is that is it necessary to do the other blocking step before incubating with other primary antibody. Thank you very much for your help.




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