Biology Reference
In-Depth Information
3. SDS-PAGE reagents are the same as described in Subheading
2.1.3.
4. Equilibration buffer I: 50 mM Tris-HCl, pH 8.8, 6 M urea,
20 % (v/v) glycerol, 2 % (w/v) SDS, and 2 % (w/v) DTT. Store
at 4 °C and temper prior to use. Add the DTT before use.
5. Equilibration buffer II: 50 mM Tris-HCl, pH 8.8, 6 M urea,
20 % (v/v) glycerol, 2 % (w/v) SDS, and 135 mM iodoacet-
amide. Store at 4 °C and temper prior to use. Add iodoacet-
amide before use.
1. Staining solution: Weigh 80 g ammonium sulfate, add 22.5 mL
of 85 % phosphoric acid, and add 700 mL of water. Dissolve
1 g of Coomassie blue G-250 in 22 mL of water. Mix the two
solutions, and then add 200 mL of methanol. Finally add water
until 1,000 mL. Store at room temperature.
2. 0.1 M Tris-H
3
PO
4
. Store at 4 °C.
3. 25 % (v/v) methanol. Store at room temperature.
4. 20 % (p/v) ammonium sulfate. Store at room temperature.
2.1.5 Colloidal
Coomassie Blue G-250
Staining
Protein identifi cation was carried out according to the protocols of
the Proteomics Service of SCAI at the University of Córdoba.
2.1.6 Protein
Identifi cation
1. Differential spots.
2. Porcine trypsin (Promega).
3. Desalting cartridges Zip Tips C18 (Agilent Technologies).
4. Peptide calibration standard (Bruker Daltonics), consisting of
a combination of peptides that provides a good calibration
across a typical mass range between 1,000 and 3,500 Da.
5. 0.1 % (v/v) trifl uoroacetic acid (TFA).
6. Ammonium bicarbonate.
7. Acetonitrile (ACN).
8. Trifl uoroacetic acid (TFA).
9.
-cyano hydroxycinnamic acid.
10. MALDI mass spectrometry calibration standards.
11. Matrix solution: 4.7 mg/mL
α
α
-cyano-4-hydroxycinnamic acid
(Sigma) in 70 % (v/v) ACN.
12. AnchorChip MALDI target (Bruker Daltonics).
2.2 Equipment
and Software
1. Airtight polyethylene bags.
2. A knife.
3. Blade mill (Moulinex AD56 42).
4. Microsieve (Ø: 15 cm, 1 mm).
