Biology Reference
In-Depth Information
-glycerophosphate, 1 mM
Na-pyrophosphate and one tablet of protease inhibitor
Complete Mini-EDTA-free (Roche Diagnostics) per 10 mL
buffer.
2. Immobilized pH gradient (IPG) buffer: IPG buffer pH 4-7
and IPG buffer pH 3-11 NL (GE Healthcare).
3. IPG strips pH 4-7 and pH 3-11.
4. Ultra-clear ultracentrifugation tube (344062, Beckman
coulter).
5. SW60Ti rotor and ultracentrifuge.
Na
3
VO
4
, 1 mM NaF, 2 mM
β
6. Rehydration solution: 8 M urea, 2 % (w/v) chaps, 0.5 % (v/v),
IPG buffer, 0.002 % bromophenol blue.
7. Agarose sealing solution: 0.5 % agarose NA (GE Healthcare),
0.002 % bromophenol blue stock solution in SDS running
buffer (
see
Subheading
2.3.3
). Add all reagents and carefully
heat the solution in a microwave until the agarose is completely
dissolved. Dispense 1.5 mL aliquots to be stored at 4 °C. The
solution is long-term stable. Upon use, boil and keep at 60 °C
before sealing.
8. SDS equilibration buffer solution: 6 M urea, 75 mM Tris-HCl
pH 8.8, 29.3 % (v/v) glycerol, 2 % (w/v) SDS, 0.002 % bro-
mophenol blue. This is just a stock solution, upon use prepare
(a) SDS equilibration buffer supplemented with 0.04 mM
DTT and (b) SDS equilibration buffer supplemented with
0.25 g IAA per 10 mL buffer.
9. Mineral oil/Dry Strip cover fl uid.
10. Solutions 3-5 specifi ed above for
1D SDS
-
PAGE separation
.
11. Immobiline rehydration tray.
12. Ettan IGPhor isoelectrical focusing unit (or similar
equipment).
1. Trypsin (or other protease, e.g., LysC or chymotrypsin).
Enzymes are provided as powders. Dissolve in 1 mM HCl at a
concentration of 1
2.3.5 In-Gel Digestion
(Suitable for Excised
Pieces from Both 1D
and 2D SDS-PAGE)
μ
g/
μ
L. Aliquot 1
μ
g/vial and store at
−20 °C.
2. 50 mM NH
4
HCO
3
: Prepare fresh from stock solution of 1 M.
3. Acetonitrile.
1. Loading buffer A: 0.4 % TFA, 80 % acetonitrile, and 19.6 %
Milli-Q water.
2. Loading buffer B: 25 % lactic acid, 60 % acetonitrile, 0.3 %
TFA, 14.7 % Milli-Q water.
2.3.6 Phosphopeptide
Titanium Dioxide
Enrichment
3. Elution buffer A: 5 % ammonia solution and 95 % Milli-Q
water.
