My advice is to do those specific steps manually off-line. I use the MCA -
Dpa resonance
transfer pair more than Edans - Dabcyl. However, I have found that I come
out ahead doing
everything I can to maximize the incorporation of my key residues and it is
essential to cap
after incorporation of the quenching species, since continuing unquenched
chains raises the
background. Even minute amounts of unquenched chains increases the
background so you need a forced cap. The on-line test for whether to cap or
not is not sensitive enough in this case. Many people double couple -- even
then, you should cap. Also, you may find steric hindrance for several
residues immediately following some of these special residues, especially
when NO2 groups are involved (as in Dpa, nitro-Tyr, etc.). I program extra
coupling and deprotection time in those modules from the outset by adding I
(vortex and wait) modules to the cycles. Note, I add the Function 2 "vortex"
to the wait module so that mixing continues.
Unprotected OH residues get acetylated if you cap after they are added.
Acetylation on ring OH
is variable. With one cap cycle after the addition, you will probably see 30
- 50% acetylation.
Hope this helps.
Susan C. Howard
susan.c.howard@monsanto.com
-----Original Message-----
From: PaxtonR@immunex.com [mailto:PaxtonR@immunex.com]
Sent: Monday, January 10, 2000 7:19 PM
To: Recipients of ABRF List
Subject: PepSyn: fluorescently labeled peptides
We are interested in synthesizing a peptide on an ABI 433A for use in a
fluorescent quenching assay, and we have a few specific questions:
1. Are there any solubility problems with using cartridges loaded with
FMOC-Abz-OH, FMOC-Tyr(m-NO2)-OH, FMOC-Lys(DABCYL)-OH, or FMOC-Asp(EDANS)
-OH?
2. Are there any problems associated with the unprotected hydroxyl on the
side chain of FMOC-Tyr(m-NO2)-OH?
3. Does the use of any of these compounds in peptides require that the
cleavage conditions be modified?
Thanks for any help.
Ray Paxton
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