Kay Finn
last updated: 4/1/02
We need to ask to use the Baker Lab FPLC. Check with Renee and Becky and Ana
before planning a run. We have found it is best to re-launch the software to
make sure every part of the FPLC is communicating well.
Our Column is on valve #8 on the Baker Lab FPLC system. We share this valve
with other columns, so re-connect it to the former column when finished. All
runs have been done as Manual runs from the ‘Kay’ directory. If you
do a run from another directory, we can move your files later.
Presently we are using a Source 15Q 10/10 column. (We also have a Mono Q.)
Buffer A = 50 mM Tris pH 8.8
Buffer B = 1M NaCl in 50 mM Tris pH 8.8
Buffers are filtered, degassed, and stored at 4ºC.
WASH OUT LINES:
Initially start up the Source 15Q column by washing out the lines at 4 ml/min,
50%B, injector set on load, valve set on Bypass, 5 minutes end time, with water
in both A & B lines.
PRIME LINES:
Place lines for Buffers A&B into reservoirs. Prime lines with 50% B, injector
set on load, Valve set on bypass, 2 minutes end time should be enough at 4 ml/min.
CONDITION COLUMN:
Run at least 10 ml of buffer 100%B on the column. 4 ml/min, injector set at
load, valve #8, 3 min.
Then, wash out salt with 100% A, 4 ml/min, injector set on load, Valve set #8,
10 minutes end time.
INJECT SAMPLE:
Loops of various sizes are available: 500ul, 1ml, 2ml, and the loops can be
connected in series to add up to 5.5 ml. To inject with loops and syringe:
Stop flow, put injector setting on LOAD, using syringe, wash loop with
buffer (5x loop vol.) Set injection valve to INJECT and remove syringe.
This prevents air entering the loop and/or buffer draining out.
Fill syringe with sample: up to or equal 1/2 total loop volume.
Insert syringe in port on injection valve, set valve to position LOAD.
Load sample into loop, leave syringe in position.
Sample gets onto column when INJECT is selected as position in method,
or manually selected.
To load larger samples: Samples can be pumped onto the column using the A buffer
line, if the pressure is watched and any overflow is captured in case of errors
in valve position or overloading. A buffer line is rinsed with A buffer and
this is pumped on the column as well: 4ml/min, valve position #8, set end time
to volume.
Once sample is on the column, wash with A buffer to baseline (2X col vol = 20ml)
and then start the gradient. We have been doing 0%B to 30%B over 1hr. 4ml/min,
injection valve #8, fractions of 8ml to start then down to 2ml when we see our
peaks start to appear. Just change the fraction settings (do not restart the
gradient). We have stopped before the run gets to the 1 hr, after peaks elute
and absorbance returns to baseline. Be sure to watch that the fractions are
being collected!
Print report!
To clean up the column, run
the gradient up to 100% B to clean resin bed. Leave here until next run.
Wash lines out on 50% B, with water in A & B resevoirs, 4 ml/min, valve
position set on bypass, then pump and store in 20% EtOH if no one is waiting
for the FPLC.
To store column if finished for over 1 week: equilibrate column in 20% EtOH/H2O.