We have used both FITC and fluorescein-free acid (Sigma F7505) for peptide
labelling. The free acid form does take a little longer to couple (8 hr-O/N),
but the cost difference (including the need for the additional amino acid) made
it our first choice. If you can pass the additional reagent cost on to the end
user, the FITC method would probably be better. I've also cleaved and purified
an NBD labelled peptide. The label was stable to TFA cleavage, but I don't have
any synthetic details for you.
Jeni
> David,
>
> I don't have any experience with the NBD group, but you can label the
> N-terminus with FITC (or any other isothiocyanate), provided you first
> attach a non-alpha amino acid (e.g., beta-alanine, 6-aminohexanoic acid,
> etc.) linker to the N-terminus. Otherwise during the TFA cleavage step,
> the labeled amino acid will undergo Edman degradation and you will lose the
> label. Fluorescein is stable to the cleavage conditions. We have used
> this strategy a number of times.
>
> Richard Laursen
>
> >For the labelling experts:
> >A customer has requested a peptide in two versions labeled at the N-terminus
> >with either fluorescein or NBD (7-nitrobenzo-2-oxa-1,3-diazole). I have two
> >questions:
> >
> >1. In your experience, which is the best way to couple each fluorophore? The
> >customer suggests the isothiacyanate (FITC and NBD-ITC, respectively). Is
> any
> >of you familiar with those or can you suggest a better alternative?
> >
> >2. What kind of acid stability do these labels have? We can use either Boc
> >(HF) or Fmoc (TFA) chemistry, but were successful with Boc chemistry before
> >with the unlabelled peptide. Any advice on that?
> >
> >I am aware that similar questions have been asked before from the relatively
> >few experts in the list. I am sorry for the reiteration. Thanks in advance
> for
> >your advice. Best regards,
> >David Andreu
> >Universidad de Barcelona
> >Depto. Quimica Organica
> >Tel/Fax: 402 1260
> >E-mail: andreu@admin.qo.ub.es
>
> Richard A. Laursen
> Department of Chemistry
> Boston University
> 590 Commonwealth Ave.
> Boston, MA 02215
> (617) 353-2491 (office)
> (617) 353-2485 (lab)
> (617) 353-6466 (FAX)
> laursen@bu.edu
>
>
> .
Janelle Lauer
Florida Atlantic University
Department of Chemistry and Biochemistry
jlauer@fau.edu
Phone: (561)297-2094
FAX: (561)297-2759