antibody cocktails - (Mar/08/2007 )

If you're setting up colocalization studies, is there any advantage to using cocktails (primaries and secondaries) aside from obviously saving time, as opposed to sequential incubation? How would you confirm if there's any steric hindrance i.e., one antibody is affecting the binding of the other?

Thanks for any input.

casandra

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you're heading into tricky ground here
all i can say is that you will get inferior results with any combination of avidin/biotin or direct labelling systems if you try combining them in the tube rather that sequentially.
if you then try to combine primaries or secondaries you will get serious cross reactivity.
even totally unrelated ones can bind to each other.

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I would have thought that intuitive but we have some reference publications using cocktails....we could always test and see but our supply of mabs are very limited and low-titered to boot...anyhow...which software do you use for the colocalization analysis? Is that tricky ground too? Too bad I'm just a few nanometers above a technically-challenged computer illiterate . Thanks Dominic.

casandra

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volocity seems to be the software of choice but then you enter the arena of deconvolution and migranes (it a nightmare to work with - maybe in a few years i'll actually get used to it).
as for the publications you mentioned i spent a year trying to produce a system for five to six antibody labels you could do in a day and it turns out the previous writers didnt do their homework - there is serious crossover which can hide itself in your choice of antibodies - for now i'm afraid i can only recomend you do it sequentially - but watch this space.

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Five to six altogether? I'm just imagining the logistics if you do them all sequentially (plus all the washes). Thanks, Dominic.

casandra

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