Western

Chris LaRosa clarosa at biocomp.unl.edu
Wed Aug 2 09:03:55 EST 2000



> 3. you need a detection system. Chemiluminiscence is most commonly used
> nowadays. Colorimetric detection (eg. BCIP/NBT) is also common but is a
> bit old-fashioned and has several drawbacks.
> 
> In your case, use the rabbit-anti-your-protein primary in combination
> with a peroxidase-coupled anti-rabbit secondary antibody (e.g. from
> Sigma) and ECL detection (Amersham). (no affiliations to either company!)
> 
> In addition, a low signal may have many reasons:
> 
> * low amount of antigen (low-abundance proteins like transcription factors)
> * low antibody quality, or too high dilution of the antibody
> * low detection sensitivity (use enhanced chemiluminiscence ECL, or a
> even more sensitive kit from Pierce)
> * too short incubations (no shorter than 1h for primary, 0.5h for secondary)
> 
> Frank

I have used BCIP/NBT westerns for years. Can you elaborate on the
advantages of these chemiluniescence systems.   If they are most
commonly used there must be a reason..... Better quality images??






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