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.
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Posted 16 November 2009 - 07:35 PM
whitebanana, on Nov 16 2009, 10:27 PM, said:
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.
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
