Pink fluorescein??

simon sbe at biochem.usyd.edu.au
Wed Mar 13 03:26:52 EST 1996


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
-- 
_______________________________________________________________
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
_______________________________________________________________
Every complex question has a simple, straight-forward,
easy to understand, wrong, answer.
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