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immunofluorescence and western blotting


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#1 whitebanana

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Posted 16 November 2009 - 06:24 PM

My dear seniors,

Can someone please tell me what ALL are the scientific reasons when Immunofluorescence staining is positive and Western blotting negative.
Ofcourse what I see in immunofluorescence staining is positive and not any unspecific staining. shRNA knockdown has confirmed this.
where i can read the scientific reasons for this case.

#2 miBunny

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Posted 16 November 2009 - 06:35 PM

Are you using the same antibody for both? Some antibodies recognize the native form of the protein (such as in IHC) but not the denatured form of the protein (such as a western blot where SDS/detergents, reducing agents, and boiling have linearized the protein) and vice versa.

Did your western work? Do you get positive results with a different antibody for a different protien?

Does your protein solubilize with your lysis conditions? Could it have precipitated out (a problem with some of the multipass membrane proteins)?

I doubt this is all the reasons but it is what I could come up with off the top of my head and wouldn't you rather have some ideas than none :P

#3 whitebanana

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Posted 16 November 2009 - 06:54 PM

Are you using the same antibody for both? Some antibodies recognize the native form of the protein (such as in IHC) but not the denatured form of the protein (such as a western blot where SDS/detergents, reducing agents, and boiling have linearized the protein) and vice versa.

Did your western work? Do you get positive results with a different antibody for a different protien?

Does your protein solubilize with your lysis conditions? Could it have precipitated out (a problem with some of the multipass membrane proteins)?

I doubt this is all the reasons but it is what I could come up with off the top of my head and wouldn't you rather have some ideas than none :P


Yes I use the same antibody, and I am doing immunofluorescence staining cells and not IHC.
you say about native form of protein, when i treat with 4% of PFA for fixation for my cells that also denatures the protein right

yes western work with other antibody with for the same protein and do solubolize with lysis buffer.

with what scientific points ( theoretically) I can argue or defend my finding when i have this result immunofluorescence positive and western negative

#4 miBunny

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Posted 16 November 2009 - 07:10 PM

paraformaldehyde cross-links proteins, it doesn't linearize them (like a western). Some antibodies recognize the 3-d conformation of the epitope, some recognized the linear (2-D) form of the epitope, and some recognize both. Is the antibody a monoclonal or a polyclonal? Polys are generally more "forgiving" about epitope recognition.

Sounds like your western blot/transfer is working well.

You can check with the antibody manufacturer. They may have an answer to the native/denatured question of epitope recognition.

Are there any different antibodies available that are validated for western blotting?




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