Re: peptide sequencing

ABRF (abrf@its.caltech.edu)
Tue, 15 Jun 1999 08:55:33 -0700

At 3:33 PM -0700 6/14/99, Jeni Lauer-Fields wrote:

>Basically, the problem is one of repetitive yield. The yields look good up to
>about 5 residues and then the data falls off into nothingness (if that's a
>word).

Jeni,
It certainly sounds like a coupling problem and you are running out of
vapor phase after a few cycles because the liquid phase is too dilute to
replace the vapor phase quickly enough as cycles proceed. You might
consider switching to a new bottle at the first sign of drop off. That way
you'll be sure what corrected the problem. We use PE's N-methylpiperidine
concoction. You can test the column by running 10 cycles of PTH stds to see
if the column is holding up. Also, you may wish to change your A buffer in
case peroxides may be building up from the THF, but in that case usually
all cycles will be bad and not just the later ones.
Best of luck,