We need a little help from the sequecing gurus out there. I am a relative novice
at this so be gentle with me. Our lab has been running a 477 for 8+ years with
very little difficulty. We recently had to leave our tried and true instrument
behind and bought another one. We've never really gotten the "new" one to
perform to the standards of our old instrument and I don't know why.
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). We use all of the solvents directly from PE except for R2. We've tried an
R2 recipe from another ABRFer that's made up of DIEA/H2O/MeOH. PE is trying to
tell us that we have a bad column and we need to change all of our reagents
(especially the R2). The column is older, but the separation is still great. I
actually tried a different column at PE's request, but found it was worse than
the older one so I switched back. To be fair abour the R2, this is a recent
switch. We've had inadequate r.y. long before we switched from TMA to the
homemade solvent. It's also been suggested to us that we're applying the sample
incorrectly (even though we've done it the same way for 8 years and the problems
are more recent) and that we shouldn't sequence peptides directly on the resin
(even though we've done that for 8 years also).
I don't know sequencing well enough to know where to look for the problem. I've
changed all of the solvents and I don't feel like doing it again without a good
reason. I really think it's due to something in the cycle optimization, but I
don't know what to change first (or second...).
I know this is a long message and the solution will probably not be an easy fix,
but we really need some help.
Thank you in advance for your assistance.
Jeni
Janelle Lauer-Fields
Department of Chemistry & Biochemistry
Florida Atlantic University
777 Glades Road
Boca Raton, Florida 33431
561-297-2094/Fax 561-297-2759