Pouring the PC column:
Solution preps:
500 ml 0.5 M NaOH
500 ml 0.5 M HCl
2L 5X column buffer:
0.5 M Pipes
302.4g
10 mM EGTA
7.61g
5 mM MgCl22.04ml (4.09M)
10 mM DTT
3.1g
4L 1X column buffer make from 5X
1)
Weigh out 30 g dry P-11 resin. Add to 500 ml 0.5 M NaOH in a 2 L beaker.
Stir gently with a glass bar. Let sit for 30 min at RT. Asperate the supernatant.
Add Milli-Q water to fill the beaker, wait until the resin settle and then asperate
the water. Repeat until the pH comes to about 8. (this takes about 3-4 changes of
water)
2)
Add 500 ml 0.5 M HCl let sit at RT for 30 min. do the above water wash
until pH comes to about 5.
3)
Add 400 ml of 5X column buffer. Measure pH and titrate to pH6.9 if
needed. Wait until resin settles. Asperate off the supernatant. Wash the resin
with approximately 1.5L of 1X column buffer (no GTP).
4)
Add 500 ml 3M NaCl in column buffer to the settled resin. Let sit for 10
min. Asperate the sup. Add 1L column buffer. Transfer the suspension to a
cylinder, let settle and measure the volume. Asperate the sup. Add more 1X
column buffer to make a 70% slurry and pour into CK? column. Pump at top
speed using peristaltic pump (it gives about 5ml/min) until 1L of 1X buffer goes
through the column. Then Pump 1L 1X column buffer with 0.1 mM GTP
through the column.