Can someone tell me if in the detection phase of the WB I can use a fluorochromo such as FITC or Sybr green instead of the kit with ECL detection on film? The visualization of the bands would direct the membrane with CCD camera. The dilution of the DMSO Sybr green affect the nitrocellulose membrane, a very aggressive. What to do?
Dear Tiane
Using flurochromes is not recommendable because of price of secondary antibodies and quentching of signal..
If you have acces to scanner like imaging device such as LI-COR Odyssey then infra red labeld secondary antibodies 700 or 800 nm are the best option because of possibility of sensitive multiple antibodies detection.
If you working with CCD cameras, Alkaline phosphatase based detection is suprior to any affordable other detection method available now.
Good Luck
It depends on your detection system recorder - but if you have the technology for Sybr green PCR then you can probably use that recorder - but with a fuorochrome-labelled antibody (not Sybr green). Probably a secondary antibody - anti-species IgG to your primary - so you get some amplification, as long as the blot is effectively blocked. A quick Google search showed a system by Advansta - but any of the new, non-fading fluorochromes (e.g. Alexa 488 instead of FITC) might do. I know from experience that ECL/film is fiddly - and we now have a fluoresence/camera system for PCR that I would be tempted to use for WB if I were to do any. Since most primary antibodies are mouse monoclonals, a secondary (say, fluorescent goat-anti-mouse-IgG) could be used on all your primaries, so save costs. If you want a two-colour system then you can use different (mouse or rabbit) primaries with species-specific secondaries with different fluorochomes.
hi,
If I'm attending using streptavidin-FITC conjugate on western blot membrane, what is the best\quickest method to witness binding?
(Important to mention that in this stage I'm only looking for absence/presence of my biotin labeled protein and not quantifying it)
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