Dear Michael:
The coupling mix we mostly use for on-resin labeling is:
FITC (20fold molar excess) in: 0.05M borate pH 9.0, ethanol, DMF
(1:1:8; by volume).
Add it to the resin, couple 24h while shaking and check by Ninhydrin
test (sometimes the resin beads are red, then the ninhydrin test is
not reliable) and mass spec.
This and similar protocols for other fluorescent dyes is published
in: Alberto Chersi et al. Biochimica Biophysica Acta 1336 (1997)
83-88.
In case this procedure does not yield complete coupling, we use 5,6
carboxyfluorescein (5 fold molar excess) for the second attempt.
Coupling with HATU, HOAt, DIEA (1:1:2, 5 fold molar excess) in DMF
24 h.
It works for many but not for all peptides.
Fluorescein succinimidester probably works great as well, but I never
tried it since it is very expensive.
Good luck!
Henriette
>Would anyone with experience attaching dyes such as fluorescein etc. to
>peptides at the N terminus post some suggestions as to the easiest,
>cheapest, least problematic methods. References and reagent suppliers
>would also be helpful.
>Thanks in advance
>
>Michael
Henriette A. Remmer, Ph.D
Director of Protein Services
Protein Sciences Facility at the Biotechnology Center
University of Illinois
311/315 Noyes Laboratory, Box 62-1
505 S. Mathews Ave.
Urbana,IL 61801
Tel: (217) 333-4695 (Lab) (217) 333-3841 (office)
Fax: (217) 244-1142
homepage: http://www.life.uiuc.edu/biotech/protein_sc.html
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