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IFS is positive and WB negative


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#1 whitebanana

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Posted 16 November 2009 - 06:27 PM

My dear seniors,

Can someone please tell me what ALL are the scientific reasons when Immunofluorescence staining (in cells) 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 jah

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Posted 16 November 2009 - 07:35 PM

My dear seniors,

Can someone please tell me what ALL are the scientific reasons when Immunofluorescence staining (in cells) 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.

I don't know about ALL, but here are a couple of potential things that come to mind when I trouble-shoot a WB.

1) Titrate your 1' & 2' antibody dilution against a positive and negative controls.
2) Are you confident your WB went well? -Electrophoresis, transfer, blocking, detection can easily go awry
3) Is your antibody sensitive to the epitope denaturation and/or reduction?


ABcam also has a nice troubleshooting algorithm




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