Susan, HI,
I got this protocol from Dick Cook--I'm forwarding a few of his
suggestions--taken from other sources and well-credited!--try the DTT
cleaning of the valve blocks--that vastly improved performance of my old
470.
M.I.T. Biopolymers Laboratory E17-415
Model 477 SEQUENCER CLEANING PROCEDURE
(from Tempst , 1994 Methods Enz.)
rev. 11/18/94
Acid cleaning of E block, reaction chamber, conversion flask , loop,and
restrictor line
1. Remove filter and install a new cartridge seal.
2. Replace S2 with neat acetonitrile.
Open valves 8,10,15,37,38 until flask is 3/4 full
CNV function 22 for 30", then CNV 20 to waste
Open valves 8,10,15,37,38 until flask is 3/4 full
CNV 22 for 30"
CNV 15 until last drop exits the restrictor
3. Replace S2 with neat TFA.
Open valves 8,10,15,37,38 until flask is 3/4 full
CNV function 22 for 30", then CNV 20 to waste
Open valves 8,10,15,37,38 until flask is 3/4 full
CNV 15 until last drop exits the restrictor
4. Replace S2 with neat acetonitrile.
Open valves 8,10,15,37,38 until flask is 3/4 full
CNV function 22 for 30", then CNV 20 to waste
Open valves 8,10,15,37,38 until flask is 3/4 full
CNV 15 until last drop exits the restrictor
CNV 13 and RXN 24 for 15", then CNV 20 for 30"
5. Loop loading
CNV 12 for 12", then CNV 22 for 4"
CNV 12 for 12", then CNV 22 for 4"
CNV 15 (optimize load injector time)
6. Remove S2 bottle
Open valves 8,9 for 30"
7. Install new ethyl acetate on S2.
RXN 17 for 120", then RXN 29 for 240"
----------------------------------------------------------------------------
--------------------------------------
This procedure can be modified as follows: Allow TFA to sit in
valve blocks (A,E), etc for up to 30' (wait for TFA to exit into waste
container to assure path is filled), followed by a 5' argon dry. 25% TFA is
then put in S2 and the pathway is again filled. 25% TFA is allowed to sit
for 2-4 hours, followed by a 5' argon dry. Backflush the S2 line and fill
replace S2 with ethyl acetate. Deliver ethyl acetate through the path for
10' and follow with a 5' argon dry
Other Routine Maintanence
DTT WASH OF VALVE BLOCKS done monthly
Take 4 mg of DTT and dissolve in 1cc EtAc. Add 100ul to 200 cc of S1 and to
S2. (0.0002% DTT in S1 and S2). Run the sequencer until RY increases and
then remove the DTT solvents and replace with unadulterated solvents.
Several DTT artifact peaks may arise on the profile- definitely one will
arise between pro and met. Pro artifact related to % thf in A buffer. Lower
[THF] makes it elute earlier (see Andre Dauphene and Bob Bates)
benefits:
Improves recovery of Trp, Lys and increases RY to 95%
Sometimes RY will be low regardless of efforts to tune the instrument.
Sometimes a bad E block is responsible but other times the cause is buildup
in the valve blocks of something that keeps RY low and may also reduce
recovery of Lys and other basic residues by 70% or more. Whatever causes
oxidation gets removed or reduced.
DTT can also be added to S3 but S3 is used in ABI cycles to transfer the
ATZ to the conversion flask so putting DTT in S3 will add a lot of DDT to
the HPLC and cause severe artifacts. Recommend not putting in S3 because
there is DTT in R4 that sees the conv. flask anyway. There is 01% DTT IN
R4.
Things more subject to oxidation imed before tfa added to form cylization!
Its good to have it in S1 S2 profilactically if you can live with the pro
artifact peak..
Other cleaning techniques:
See Techniques in Protein Chemistry IV, p. 410 Donna Atherton, et al. 1994
Check vent line path from flask
PTH Chromatogram Fine Tuning
Adding 1% acetone to A buffer will lower the end of the smile profile and
also allow the auto zero point to be set at a lower latitude.
If Asp is too close to DTT, add 100ul glacial HOAC to 1liter of A3 (can
also use 100ul of neat TFA (note , tried tfa 11/15/94 and saw tremendous
downward drift- need to be at .01o scale to use TFA). This will retard ASP
but also GLU. If GLU is not seperated from DMPTU, increase the 0.6 time 5B
to 14%. Use ultra high purity HOAC to avoid artifacts. [this procedure was
highly unsatisfactory when tried in 1995 due to falling baseline. Perhaps
it could work if no acetone was added to A]
Seperating Glu from DMPTU: Increase the %B to about 12 or 14 at 0.6 min.
to seperate
separate the cys-propionamide from DMPTU by playing with the initial
percent B in the gradient. (formed when the gels are not completely
polymerized. The free acrylamide S-alkylates resulting in
PTH-(S-propionamide)-cysteine
Use 0.3mm or larger teflon tubing for restriction line on model 120
Replacing solvent frits, especially the A frit, can reduce baseline noise.
A frit must be replaced if air bubbles are seen in lines as pump is
refilling.
The peak imediately prior to asp is ozidized DTT. If D is too high on the
oxidized DTT shoulder, get better R4 with a greater DTT/oxidized DTT ratio.
Better DTT will improve Lys yields too.
If hydrophobic residues are low relative to hydrophilic residues or if the
standard profile peaks are lower than the normal profile peaks. check to
see if there is a precipitate at the botton 1/8th inch of the pickup tube.
This material is from the ABI filters and can not only clog the
transfer/loading system but also be responsible for seemingly unexplainable
differences in yields both intra and inter cycle.
>Hello dear all!
> My first message for you in the new millenium !
>In one of our 477¥s, I am having very low repetitive yield, especially for
>peptides.
>I checked the S2 delivery, ( it is now lower than the lowest level
>recommended by the optimization rules ), I changed the TMA ( the RY improved
>a little ) and also, I ran programs with different extraction conditions (
>less and more post-TFA argon dry times ) but no differences were obtained,
>eventhough these parameters are supposed not to have any influence in the
>RY. What may gives any clue about, is that the glutamine, in
>B-lactoglobulin, shows a some high deamidation effect ( about half of the
>glutamine peak ).
>This situation is the same, even with different reagents lotes.
> Any suggestion would be welcome.
>Thanks in advance for your help.
> Happy 2000 !
>
>********************************************
>Susana Linskens
>LANAIS-PRO
>Junin 956
>1113 Buenos Aires - ARGENTINA
>linskens@qb.ffyb.uba.ar
>FAX: 54-11-4508-3652
>Phone: 54-11-4508-3651
>********************************************
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