RE: PepSyn:Problems labeling with Fluorescein

Holyst, Trudy (tholyst@bcsew.edu)
Wed, 15 Sep 1999 13:17:22 -0500

Hi Lisa, I am partial to using carboxy fluorescein from Molecular Probes.
You can get the 5,6 carboxy as the mixed isomer or the single isomer form. I
incubate the peptide resin with the fluorescein in neat DMF overnight at
room temp on a nutator. I use a 5 ml tube with app 2 mls of solvent in it;
the resin should be well solvated but there has to be enough headspace to
insure good mixing. I use roughly the same umol concentration of resin to
label. I also add DMAP at a concentration of 30% excess of label. I transfer
the resin to those great little syringe cloumns Gilson used for the multiple
peptide machine, do a solvent exchange with DCM, dry on the lyophilizer and
cleave as normal. This has worked for me on lots of peptides.

Trudy Holyst

Peptide Core
Blood Research Institute
Blood Center of SE WI
Milwaukee, WI 53201

mtholyst@BCSEW.edu

-----Original Message-----
From: lisa bibbs [mailto:bibbs@scripps.edu]
Sent: Tuesday, September 14, 1999 4:25 PM
To: Recipients of ABRF List
Subject: PepSyn:Problems labeling with Fluorescein

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?