photo bleaching - (Mar/23/2006 )

hi bioforum members,
does any one help me in understanding the exact mechanism behind photo bleaching in western blotting?
given situation
1, lot of protein is present on transferred membrane
2, when we use low dilution of primary and secondary antiobody (high conc), we see so called donuts where detected protein band is whilte in center and dark in periphery (quite common in alpha tubulin detection).
i know that it is photo bleaching but i m wondering what could be the exact mechanism?

thanks in advance

gud luck for ur research

payeli

I'm not sure if this is correct, but let me try...

this occurs when the system is saturated...REALLY saturated. when the detection reagents are added to the membrane at the end, there is so much of everything present that the chemi reaction is basically like a flash or tiny explosion of light...this shows up on the film as a halo or a ghost band

hi aimikins,
thanks for ur reply, in ur opinion system saturation means, the film has certain reactivity range, when there is heavy signal it can not be anymore sensitive to light, because of this reason film shows this ghost band.
is it so?
payeli

honestly, I'm not sure, but your theory makes the most sense

hey, for some good information, check out this link

hi aimkins,
yup, these people(Pierce article) also have same porblem and they did trouble shooting. moreover they could not explain the porblem in our discussion, but they give precautions to avoid gohst bands.
to understand the phenomena, i went through google on photography and developement,
in one instance they give the explanation whihc i was discussing with u in my last post. but the difference was they meant printing films, so i just extrapolated those observation in context to our western blotting.
let me see if any of our group members will give even more sensible answer (if there is any).

thanks