[Histonet] streptavidin-alexafluor conjugates

[PALMER Jason (SVHM)]

Hi all.

Just wondering what sort of experiences people have had with streptavidin-alexa fluor conjugates, and in particular AF350? (We want a blue dye conjugate to use in a double labelling procedure on tissue which has GFP labelled cells; CY-3 is used to detect the other Ab).  I tried this reagent recently, at a range of dilutions, substituting it for a streptavidin-HRP label with an antibody and method that always works well on FFPE tissue, but got absolutely no signal.  I do intend to try it again, but thought a histonet query might be a good idea at this point.  I did come across a technical note (7.4, p 299 10th edition Mol Probes handbook) stating that "fluorophores conjugated to avidin and streptavidin may be quenched significantly, apparently because the dyes interact with amino acid residues in the biotin binding pocket...".  It goes on to say that the addition of free biotin can help recover some of this lost signal.  Does anyone have a comment or experience on such quenching, or tried free biotin blocking?

Any comments or suggestions gratefully received, as always!

Jason

Jason Palmer
Histology Laboratory Coordinator
Bernard O'Brien Institute
42 Fitzroy St, Fitzroy Victoria 3065
Australia
tel +61 3 9288 4018
fax +61 3 9416 0926
email: jason.palmer <@t> svhm.org.au

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