Buffer choice and strength depends on your sample's solubility and
down-stream clean-up procedures. You can get by with any buffer that
maintains the pH around 7.5, and maintains the solubility of your sample and
the iodofluorescein. If your concerned about metal catalyzed oxidations,
then add EDTA to your reaction at around 10 mM, sparging your buffer with Ar
would not hurt either. If your sample is stable, then run the reaction at
room temp., the reaction will go much faster. I have not had the best luck
using dialysis to clean-up samples that have been conjugated with
fluorescein or other fluorescent derivatives; the unconjugated Flour. tends
to stick to the dialysis membrane, also, fluorescein and related structures
can be hydrophobic and do not dialyse very well. If you have an LC method
for clean-up use it instead. In general, a good starting point is to
conjugate at a ratio of 2 to 5:1 label to peptide. This reaction is pretty
specific, so you should not have a problem of labeling at other sites.
Also, as you mentioned run this reaction in the dark. There should not be a
need to quench the reaction if you clean-up the sample soon after labeling.
Hope the advice helps.
Rob
-----Original Message-----
From: Angela c. Murphy [mailto:acmurphy@helix.nih.gov]
Sent: Monday, June 26, 2000 7:00 PM
To: Recipients of ABRF List
Subject: Coupling of iodoacetamidofluorescein
I will be coupling 5-iodacetamidofluorescein to two different peptides,
one a 10-mer and the other a 26-mer through the -SH of cysteine. The
reference I have says the coupling was done in pH 7.5 sodium phosphate
buffer/acetonitrile 1:1. It does not give the molarity of the phosphate
buffer and does not mention if EDTA was present or if Argon was bubbled
through the buffer. It also does not mention what, if any, molar excess
of fluorescein over peptide was used.
I've checked other references, and found a variety of buffers used, TES,
100 mM ammonium bicarbonate, etc., and various molar excesses - 1.2 -
3-fold. Some folks react at room temp., others at 4 degrees C. Some add
DTT or mercaptoethanol to quench the reaction, others dialyze or do HPLC.
Since this is a first for me, I would be extremely grateful if anyone had
any suggestions for insuring the success of the coupling reaction. I do
intend to do the reaction in the dark, and to follow it by HPLC. I will
purify the product by HPLC - 6 mM HCl/acetonitrile. (Both peptides will
be injected into cells, and TFA is a no-no.)
Thanks in advance for any and all help.
Angela C. Murphy
*******************************************
Angela C. Murphy, Chemist
Lab. of Cell Biology, NHLBI, NIH
3 Center Drive, MSC 0301, Rm. B1-22
9000 Rockville Pike
Bethesda, MD 20892-0301 USA
tel.: (301) 496-2324
fax: (301) 402-1519
email: acmurphy@helix.nih.gov
*******************************************
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