Biology Reference
In-Depth Information
2.2 Isoelectric
Focusing (IEF)
1. IEF extraction/solubilization media: 8 M urea, 2 M thiourea,
2 % (w/v) CHAPS, 2 % (v/v) Triton X-100, 50 mM DTT
(
see
Note 2
).
2. Immobilized pH Gradient (IPG) buffer or Ampholytes.
3. Mineral oil.
2.3
SDS-PAGE
1. 1.5 M Tris-HCl, pH 8.8.
2. 30 % acrylamide/bisacrylamide solution (29:1).
3. 10 % APS in water.
4. TEMED.
5. Isobutanol.
6. SDS equilibration buffer: 1.5 M Tris-HCl, 6 M urea, 30 %
(v/v) glycerol, 5 % (w/v) SDS (if used stacking gel) or 6 M
urea, 50 mM Tris-HCl, pH 8.8, 2 % (w/v) SDS, 30 % (v/v)
glycerol (if stacking gel is not used) with 2 % (w/v) dithiothrei-
tol (DTT) or 2.5 % (w/v) iodoacetamide (IAA) (
see
Note 3
).
7. SDS running buffer: 25 mM Tris, 0.192 m glycine, 0.1 %
(w/v) SDS.
8. 0.5 % (w/v) agarose in SDS running buffer with traces of bro-
mophenol blue.
9. Colloidal Coomassie Blue staining solution: 20 % (v/v) etha-
nol, 1.6 % (v/v) phosphoric acid, 8 % (w/v) ammonium sul-
fate, 0.08 % (w/v) Coomassie Brilliant Blue (CBB) G-250
(
see
Note 4
).
1. Wash solution: 50 % acetonitrile, 50 mM ammonium
bicarbonate.
2. 100 % acetonitrile.
3. Trypsin in 50 mM ammonium bicarbonate.
4. Extraction solution: 60 % acetonitrile, 1 % formic acid.
2.4
Protein Digestion
3
Methods
This extraction method is modifi ed from Hurkman and Tanaka [
7
].
3.1 Phenol-Based
Protein Extraction
1. Grind sample (0.5 g) to a fi ne powder with liquid nitrogen,
mortar and pestle.
2. While still in mortar, immediately resuspend powder in 10 mL
of homogenization solution. Homogenate will freeze
(
see
Note 5
).
3. Allow homogenate to reach room temperature (RT), transfer
to a 50 mL propylene tube and mix slowly on a shaker for
30 min at 4 °C.
4. Centrifuge at 5,000 ×
g
and 4 °C for 15 min.
