On Tue, 12 Sep 2000 10:43:11 -0700 Sandy Kielland <Kielland@uvic.ca> wrote:
> 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.
Sandy, your problem can be the stock time of the column. Those PTH-columns,
when stocked for more than six months, can loose silica when used. That
silica is deposited into the detector, becoming an obstacle to the light
passage, which drastically reduces the sign of the PTH-amino acid. Give a
glance at the detector. If the window is white, it's because silica was
drained from the column. You will have to change the column as well as to
clean the detector.
On Wednesday, September 13, 2000 1:21 PM Alex Carne <sandyc@icr.ac.uk>
wrote:
> Sandy,
> I have encountered a very similar problem.All the PTHs were OK until after
Pro
> and then the peak heights dropped dramatically.Columns were changed,blocks
> cleaned,hair pulled out etc etc.If you are using ABIs B2 then this may not
> apply but I went back to adding DMPTU to B and I now have decent sized
peaks
> again.
Alex, are you using a special cycle for proline? Here in my laboratory,
whenever we know that in a certain cycle a proline is expected, we
programmed for that cycle a different reaction cycle, specific for proline.
It's a reaction cycle with prolonged coupling and cleavage times, because,
like you well knows, the proline, due to its own structural characteristics,
reacts more slowly with PITC. Otherwise, a drastic reduction in peaks
heights is expected. Unassuming that the sequence becomes out of phase.
Best regards.
-------------------------------------------------------
Prof. Ricardo B. Cunha
Brazilian Center for Protein Research and Services - CBSP
Laboratory of Protein Chemistry and Biochemistry
Institute of Chemistry
University of Brasilia
Brasilia - DF Brazil
ZIP CODE: 70910-900
Tel: 55-21-61-307-2142
Fax: 55-21-61-272-4548
mailto:rbcunha@unb.br
http://www.geocities.com/Athens/Acropolis/5221
http://www.unb.br/cbsp
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