From: Adrian Smith (A.Smith@centenary.usyd.edu.AU)
Date: Sun May 11 2003 - 19:04:47 EST
Hi all, We have a monoclonal* that we use routinely that we find very difficult to conjugate - usually what happens is the conjugates precipitate rapidly after the procedure. This can happen in a matter of hours, days or weeks. In the past we have tried it with biotin, APC and PE and have had poor results. Recently we did it again with a biotin with a long chain linker and the results were much better, i.e. better separation between postive and negative peaks and the conjugate has been stable for several months. Last week we tried a conjugation with AlexaFluor 594 (the succinimidyl ester form) and we already see the "clumps" in the mixture. The other antibody we did at the same time looks fine. We are looking for suggestions for alternative approaches that will allow us to conjugate this antibody with a fluorochrome. Does anyone know of any fluorochromes that come with a long-chain linker? (We have a lot of other antibodies that we can only obtain as biotin conjugates and the alternative, which is to do an anti-rat stain with blocking, is painfully slow, so we are very keen to get this monoclonal attached to something useful!) Thanks, Adrian Smith * the monoclonal is RR8.1 (anti-mouse TCR Va11; I believe it is a rat IgG2b) -- _________________________________________ Adrian Smith (Research Officer) T CELL BIOLOGY Centenary Institute of Cancer Medicine & Cell Biology
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