Fluorescein can be attached readily to peptides in solution by using
fluorescein isothiocyanate (FITC, Aldrich F250-2). If the peptide contains a
lysine, some preference for attachment to the alpha amino versus the lysine
side chain can be obtained by doing the coupling at pH 7.5.
If FITC is coupled to the peptide on the resin, acid treatment (HF or
TFA) to remove the peptide from the resin will cause partial or total loss
of the fluorescein and the N-terminal amino acid, since the thiourea formed
is the intermediate in the Edman degradation.
Since fluorescein (Aldrich F245-6) contains a carboxyl group, it can be
coupled in SPPS. The fluorescein carboxyl is hindered, so HATU should be
used to get good coupling.
John M. Stewart
Department of Biochemistry B126
Univ. of Colorado School of Medicine
4200 E. Ninth Ave.
Denver, CO 80262
phone: 303-315-7534
FAX: 303-315-8215
email: john.stewart@uchsc.edu
-----Original Message-----
From: Roger Murphy [mailto:Roger.Murphy@ludwig.edu.au]
Sent: Monday, February 19, 2001 2:39 PM
To: Recipients of ABRF List
Subject: RE: High Molecular Weight Proteins
Don't know about fluorescein, but biotin is a snap. Presumably you're
attaching to the N-terminal, so just treat your biotin as another amino acid
coupling. Works nicely!
Cheers,
Roger
At 03:23 PM 2/19/01 +0000, neil brew (BI) wrote:
Dear All
I have got to make some peptides labelled with biotin and
fluorescein. This will be the first time I have attmpted such labelling and
I wonder if anybody out there in ABRF-land has any Protocols/references for
on resin labelling of peptides with such labels as biotin/fluorescein/DIG
etc.
Many thanks in advance, Neil Brew
------------------------------------------------------------
Roger Murphy, Ph.D.
Biological Production Facility
Ludwig Institute for Cancer Research
Austin & Repatriation Medical Centre
Studley Road,
Heidelberg, Vic. 3084
Australia.
Tel 61-3-94965463
Fax 61-3-94965436
Email Roger.Murphy@Ludwig.edu.au
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