Biology Reference
In-Depth Information
4. Discard supernatant and repeat
step 3
.
5. Discard the supernatant and resuspend each pellet in 30 mL
ice-cold 10 % (w/v) TCA in acetone.
6. Incubate the samples in an ultrasound water bath for 10 min
at 4 °C. Add crushed ice to keep the samples cold.
7. Recentrifuge and wash each pellet twice with 30 mL ice-cold
10 % (w/v) TCA in acetone.
8. Wash each pellet once with 30 mL ice-cold 10 % (w/v) TCA
in dH
2
O and then twice with 30 mL ice-cold 80 % (v/v)
acetone.
9. Resuspend each tissue powder pellet in 24 mL freshly pre-
pared dense SDS buffer.
10. Add 24 mL tris-saturated phenol pH 8.0 and vortex vigor-
ously for ~1 min.
11. Separate phases by centrifugation at RT for 30 min at 3,000 ×
g
.
12. Transfer each upper phenolic phase to a centrifuge bottle
(phenol resistant, e.g., Nalgene
®
175 mL conical-bottom
polypropylene copolymer (PPCO) centrifuge bottles).
13. Add 5 volumes of ice-cold 100 mM ammonium acetate in
MeOH, vortex and precipitate proteins for 1 h at −20 °C.
14. Collect precipitated proteins by centrifugation at 7,500 ×
g
for
10 min at 4 °C.
15. Discard the supernatants and resuspend the protein pellets in
30 mL ice-cold 100 mM ammonium acetate in MeOH.
16. Recentrifuge protein samples at 7,500 ×
g
for 10 min at 4 °C.
17. Wash the protein samples once again with 30 mL ice-cold
100 mM ammonium acetate in MeOH and twice with 30 mL
ice-cold 80 % acetone.
18. Discard as much acetone as possible, air-dry the protein pellets
shortly on ice and dissolve each protein pellet in 6 mL IB/A
by head-over-head incubation at 10 °C overnight.
19. Alternatively, dry protein pellets can be stored at −20 °C.
3.2 Al(OH)
3
-Based
MOAC Enrichment of
Phosphoproteins
1. Pool two protein pellets each dissolved in 6 mL IB/A in a
50 mL tube.
2. Spin down undissolved proteins by centrifugation at 7,500 ×
g
for 10 min at 10 °C.
3. Transfer the supernatant to a clean 50 mL tube and determine
protein concentration by Bradford assay.
4. Adjust the protein concentration to 3 mg/mL using IB/A.
5. Transfer 12 mL of the 3 mg/mL protein solution in IB/A to
a clean 50 mL tube.
