1 reply to this topic

[whitebanana]

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.

[jah]

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.

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