I am a protein chemist/immunologist and have been trying to prepare a
derivatised aminodextran as follows:
(1) Make fluorescein-conjugated aminodextran (Mr 10000) by reacting
aminodextran with FITC. Exhaustive dialysis vs H2O.
(2) Activate fluorescein-conj.dextran using epichlorohydrin
(epichlor/NaBH4, strongly alkaline conditions - pH about 11). Dialyse
as above.
(3) Couple 1,2-diaminoethane to activated fluorescein-conj dextran.
This was done by adding diaminoethane and 0.2 M NaHCO3 to give 10%
(v/v) for each to the solution from (2).
Steps (1) and (2) yielded products with "normal" fluorescein
absorbance/fluorescence but on adding the reagents for step (3) the
typical yellow-green fluorescent solution changed to a pink colour and
has remained so even after exhaustive dialysis vs H20.
Can anybody suggest (a) what is the pink stuff and (b) how can I
modify the protocol to retain the integrity of the fluorescein.
Thanks
Simon
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Dr Simon B Easterbrook-Smith Phone: (+) 612 351 3905
Department of Biochemistry FAX: (+) 612 351 4726
University of Sydney Email: sbe at biochem.usyd.edu.au
Sydney NSW 2006
AUSTRALIA
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Every complex question has a simple, straight-forward,
easy to understand, wrong, answer.
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