Re: PepSyn:Problems labeling with Fluorescein
Jean Lucchetti (uunet!eurogentec.be!j.lucchetti@mail.uu.net)
Wed, 15 Sep 1999 12:53:29 -0700
lisa bibbs wrote:
>
> we have been trying to label the following peptide on the n-term with
> Fluorescein.
>
> Y G R K K R R Q R R R G K P P A P P M R N T S T M
>
> Before labeling, we did a partial cleavage to make sure we made the intact
> peptide and it looks great.
>
> We tried using FITC followed by washing and cleaving. We end up with ONLY
> the peptide - Y. I figure I must be going through an edman type cleavage
> during cleavage and deprotection even though we keep it pretty cool. (I
> know some are saying DAH!)
>
> We then used Fluorescein-NHS. Most of our product is unlabeled (90%) and
> then there is a product that is that is -17 amu.
>
> What is your favorite protocol for labeling with Fluorescein?
Lisa,
You can couple the 5-carboxyfluorescein ( or the cheapest
5,6-carboxyfluorescein isomer mixture but you will end with a
diastereoisomeric mixture is that case) both from Molecular Probes
directly to the NH2 term using your standard coupling procedure (
extended coupling time ). It works nicely in our hands. I was told
fluorecein itself works too, but I haven't experienced it.
Hope this helps.
--
Jean LUCCHETTI, Ph.D.
EUROGENTEC Bel S.A.
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