Biology Reference
In-Depth Information
2.2 Al(OH)
3
-Based
MOAC Enrichment of
Phosphoproteins
1. Incubation buffer B (IB/B): 30 mM MES pH 6.1, 0.25 %
(w/v) CHAPS, 8 M urea, 200 mM sodium glutamate,
200 mM potassium aspartate, 30 mM imidazole.
2. Incubation buffer C (IB/C): Mix 1 vol IB/A with 2 vol IB/B.
3. Incubation buffer 200 (IB200): 30 mM MES pH 6.1, 0.25 %
(w/v) CHAPS, 8 M urea, 200 mM sodium glutamate,
200 mM potassium aspartate, 20 mM imidazole.
4. Elution buffer (EB): 200 mM potassium pyrophosphate
pH 9.0, 8 M urea.
5. 2 % (w/v) sodium deoxycholate (DOC).
6. 100 % (w/v) TCA in dH
2
O.
7. 25 % (w/v) TCA in dH
2
O.
8. 80 % (v/v) acetone in 50 mM Tris pH 7.5 (stored at −20 °C
until use).
2.3 Phosphoprotein
Digestion and Peptide
Desalting
1. 100 mM ammonium bicarbonate (AmBiC), 8 M urea.
2. 100 mM AmBiC.
3. 20 % (v/v) acetonitrile (ACN), 100 mM AmBiC.
4. 10 % (v/v) ACN, 25 mM AmBiC.
2.4 TiO
2
-Based
MOAC Enrichment of
Phosphopeptides
1. Buffer 1 (B1): 50 % (v/v) ACN, 2.5 % (v/v) trifl uoroacetic
acid (TFA) in dH
2
O, phthalic acid-saturated (~500 mg of
phthalic acid saturates 20 mL B1).
2. Buffer 2 (B2): 50 % (v/v) ACN, 0.1 % (v/v) TFA in dH
2
O.
3. Buffer 3 (B3): 0.1 % (v/v) TFA in dH
2
O.
4. Buffer 4 (B4): 5 % (v/v) ammonia in dH
2
O.
3
Methods
3.1 Total Protein
Extraction
All steps during total protein extraction should be carried out using
ice-cold buffer solutions except for dense SDS buffer and Tris-
saturated phenol.
1. Grind 5-10 g (FW)
Arabidopsis
tissue to a fi ne powder in liq-
uid nitrogen and fi ll two prechilled 50 mL tubes up to the
17.5 mL mark. If not processed immediately after sampling,
store at −80 °C until analysis.
2. Pre-warm Tris-saturated phenol at RT and freshly prepare
dense SDS buffer.
3. Add 30 mL ice-cold acetone to each sample, vortex vigorously
to resuspend the tissue powder and spin down at 3,000 ×
g
for
5 min at 4 °C.
