Sandy,
Is your column bad, or is it your Edman chemistry? The first test is to run
a standard AA mix on the LC unit, as if you were calibrating the integrator
before a sequencing run. Are the hydrophobic ones all gone, or does your
problem only appear during sequencing?
If the problem only appears during sequencing, then I suspect that your
extraction is poorly optimized. Start with your drying times, since they're
the most likely to drift, especially if you have a new conversion flask. If
they're perfect, already, you may need to recalibrate your TFA and heptane
delivery times.
If you really do have a problem with the columns, then it's probably one of
two things. First make sure that you don't have all columns from the same
[bad] manufacturer's lot. It's rare, but it happens, especially to people
who live a long way from California. If you can rule that out, then I'll
bet you're sequencing samples from electrophoresis. Tell your sample prep
group that they need to scrub their samples better, because you're getting
SDS carryover, and it's wrecking your stationary phase.
Mark
-- . . . . . Dr. John Mark Carter MCarter@AxCellBio.com . AxCell Biosciences Corporation 826 Newtown-Yardley Road, Suite 100 Newtown, PA 18940-1721 office 267.757.1223 lab 267.757.1230 FAX 267.757.1301 www.AxCellBio.com . . Axcell Biosciences is a wholly owned subsidiary of the Cytogen Corporation. . . . . . "Ohne Analyse, keine Synthese." [Without analysis, no synthesis.] -Friedrich Engels, Anti-D¸hring, 1878-----Original Message----- From: Sandy Kielland [mailto:Kielland@uvic.ca] Sent: Tuesday, September 12, 2000 1:43 PM To: Recipients of ABRF List Subject: PTH columns
Hello all; Have any of you protein sequencing people been having trouble with short lifetimes on ABI PTH columns? Over the years I have expected these columns to last 3-4 months with careful use, but lately this has not been the case. I treat them very gently, break them in properly and run the older slower gradient to keep operating pressures low. I use ABI solvents and reagents. Under the assumption that there is something wrong with my instrument (ABI473) maybe leaking something nasty and poisoning the column, I have replaced the conversion flask and the only valve block I could find with a leak, block 1. Hasn't made a bit of difference. The symptoms are that my hydrophobic residues are disappearing. Weird, I know. I will see data something like this....
1. asp 8 picomoles 2. thr 5 " 3. pro 9 " 4. val 0 " 5. ser 3 " 6. asn 8 " 7. leu 0.2 " 8. tyr 1 "
When I say "0" for valine, I really mean "0"...not even a whisper. It's obviously being coupled and cleaved OK, but where did it go? Today I've installed yet another new column and will get beautiful data for about 3 weeks, then it will start to deteriorate again. Any hints much appreciated.
Best regards,
Sandy Kielland Victoria Protein Microchemistry Centre Department of Biochemistry and Microbiology University of Victoria Victoria B.C. CANADA phone: (250) 7218884 FAX (250) 7218855 Email Kielland@UVic.ca Webpage http://www.proteincentre.com
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