Biology Reference
In-Depth Information
6. Add 2 vol IB/B and immediately mix by head-over-head
incubation.
7. Clear protein solution by centrifugation at 7,500 ×
g
for
10 min at 10 °C.
8. Meanwhile transfer 2.88 g Al(OH)
3
to a 50 mL round-bottom
tube.
9. Resuspend in 36 mL IB/C to equilibrate the chromatography
media.
10. Spin down Al(OH)
3
for 3 min at 3,000 ×
g
and remove super-
natant using a 25 mL pipette.
11. Repeat
steps 8
-
10
.
12. Discard as much IB/C from the pelleted Al(OH)
3
as possible
using a gel-loader pipet-tip and add 36 mL cleared dissolved
protein sample.
13. Allow phosphoproteins to bind the Al(OH)
3
by head-over-
head incubation at 10 °C for 1 h.
14. Spin down by centrifugation at 3,000 ×
g
for 5 min at 10 °C.
15. Either discard non-bound protein fraction or store for further
analysis.
16. Wash Al(OH)
3
-bound phosphoproteins six times by resuspen-
sion in 36 mL IB200. Invert the tubes carefully and do not
vortex Al(OH)
3
-bound phosphoproteins. To spin down
Al(OH)
3
-bound phosphoproteins, centrifuge at 3,000 ×
g
for
5 min at 10 °C.
17. After the last washing step, remove as much IB200 as possible
using a gel-loader pipette tip.
18. Resuspend the Al(OH)
3
-bound phosphoproteins in 24 mL EB
and allow them to elute from the Al(OH)
3
for 30 min by
head-over-head incubation at RT.
19. Pellet Al(OH)
3
by centrifugation at 3,000 ×
g
for 5 min at RT.
20. Transfer supernatant containing the eluted phosphoproteins
to a clean 50 mL conical bottom tube.
21. Centrifuge samples at 7,500 ×
g
for 10 min at RT to pellet as
much Al(OH)
3
as possible.
22. Apply 12 mL supernatant to an Amicon Ultra-15 centrifugal
fi lter unit with a 10 kDa cut-off and centrifuge at 3,000 ×
g
for
30 min at RT.
23. Repeat
step 22
to concentrate protein sample to a minimal
volume of 250
L.
24. Transfer as much retentate as possible to a 1.5 mL Eppendorf
Protein LoBind
®
tube and dilute sample by adding 4 vol of
dH
2
O.
μ
