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
*******************************************
This archive was generated by hypermail 2b29 : Fri Jun 30 2000 - 07:51:55 EDT