Biology Reference
In-Depth Information
NaHCO
3
(0.95 g), made up to 100 mL. If necessary, adjust
the pH to 11.25 with NaHCO
3
or NaOH.
4. BCA Reagent B: CuSO
4
5H
2
O (0.4 g) in 10 mL of water.
Reagents A and B are stable at room temperature.
⋅
2.2 SDS-PAGE
1. Running Buffer (TGS): For a 10× stock mix 30.2 g Tris-Base,
144 g of glycine, and 1 g of SDS; add
dd
H
2
O up to 1 L.
2. Resolving gel. Amount required for 2 Mini-Protean (Bio-Rad)
gels: mix 3 mL of 40 % (w/v) acrylamide/bisacrylamide
(37.5:1), 2.5 mL 1.5 M Tris-HCl, pH 8.8, 50
L of 20 %
(w/v) SDS, and 4.5 mL of H
2
O. Mix well and degasify in
vacuo. Add 50
μ
L of TEMED for
starting the polymerization. Mix slowly by inversion before
casting the gel. Add 5 mL of acrylamide mix to each 7 cm gel
cassette. Avoid bubbles during casting and quickly cover the
acrylamide with 2-propanol. Let the gels polymerize for 1 h. If
larger gels are used, increase the volumes accordingly.
3. Stacking gel. Amount required for 2 Mini-Protean (Bio-Rad)
gels: mix 0.7 mL of 40 % (w/v) acrylamide/bisacrylamide
(37.5:1), 2.5 mL 0.5 M Tris-HCl, pH 6.8, 50
μ
L of 10 % (w/v) APS and 5
μ
L of 20 %
(w/v) SDS and 2.25 mL of H
2
O. Mix well and degasify in
vacuo. Add 50
μ
L of TEMED for
starting the polymerization. Before adding the APS and
TEMED discard the 2-propanol layer covering the resolving
gels and briefl y rinse with water. Then pour the stacking solu-
tion containing APS and TEMED, and carefully place the
comb avoiding bubbles.
4. Coomassie staining solution: 40 % (v/v) methanol, 10 % (v/v)
acetic acid, 0.1 % (w/v) Coomassie R-250, in distilled water,.
Dissolve the Coomassie in methanol and then add the other
components.
5. Coomassie destaining solution: 40 % (v/v) methanol, 2 %
(v/v) acetic acid in distilled water.
6. 5× Laemmli buffer: 20 % (w/v) SDS, 80 % (v/v) Glycerol,
20 % (v/v)
μ
L of 10 % (w/v) APS and 5
μ
-Mercaptoethanol, 0. 01 % (w/v) bromophenol
blue. Store at −20 °C. Before using make sure that SDS is com-
pletely dissolved. Heating may be necessary [
4
].
β
2.3 Protein Digestion
and Desalting
1. Trypsin Buffer: 25 mM NH
4
HCO
3
(AmBic), 10 % (v/v) ace-
tonitrile, 5 mM CaCl
2
. For preparing 1 mL mix 250
μ
L of
0.1 M AmBic, 100
μ
L of pure acetonitrile and 645
μ
L of H
2
O.
L of 1 M CaCl
2
, Calcium must be added in last place
to avoid precipitation. Prepare fresh.
2. Trypsin Solution: dilute Trypsin Sequencing Grade (Roche 11
418 475 001) to 10-12.5 ng/
Add 5
μ
L. Add 1 mL of trypsin buffer
to the vial, incubate on ice for 10 min and then pipette up and
μ
