4. Refrigerated centrifuge.
5. Protein quantification reagents/kit.
6. Protein low bind microtubes.
2.2 1-D SDS-PAGE
1. Loading buffer: 180 mM Tris–HCl (pH 6.8), 6% SDS, 15%
β-mercaptoethanol, 30% glycerol, 0.15% bromophenol blue.
Prepare 1 M stock of Tris–HCl (pH 6.8) dissolving 12.114 g
Tris in 100 mL ultrapure water and fix the pH with few drops of
HCl 1 M. Prepare 10 mL of loading buffer adding 1.8 mL of
1 M Tris–HCl, 0.6 g SDS, 1.5 mL of β-mercaptoethanol, 3 mL
of glycerol, and 0.015 g of bromophenol blue in ultrapure
water. Store at À20
C in 1 mL aliquots.
2. Running buffer: 25 mM Tris, 0.19 M glycine, 0.1% SDS. Prepare 10% SDS by dissolving 10 g in 100 mL of ultrapure water.
Prepare a 5Â buffer by dissolving 15.175 g Tris, 71.4 g glycine,
and 50 mL 10% SDS in 1 L of distilled water. Dilute 1/5 before
using.
3. Running gel: 12.5% polyacrylamide gel. Prepare 100 mL of
1.5 M Tris–HCl, pH 8.8, dissolving 18.17 g of Tris. Prepare
10% ammonium persulfate (APS), dissolving 10 g APS in
100 mL. Mix all components in the following order, stirring
after each addition: 3.175 mL of ultrapure water, 4.17 mL of
30% acrylamide/bisacrylamide solution (ratio 37.5:1), 2.5 mL
of 1.5 M Tris–HCl (pH 8.8), 100 μL of 10% SDS, 50 μL of 10%
APS (freshly prepared), and 5 μL TEMED. The last two components should be added just before using.
4. Stacking gel: 4% acrylamide gel. Prepare 0.5 M Tris–HCl
(pH 6.8) by dissolving 6.057 g Tris in 100 mL ultrapure
water and fix the pH with a few drops of HCl 1 M. Mix all
components in the following order, stirring after each addition:
3 mL of ultrapure water, 660 μL of 30% acrylamide/bisacrylamide solution (ratio 37.5:1), 1.26 mL of 0.5 M Tris–HCl
(pH 6.8), 50 μL of 10% SDS, 25 μL of 10% APS, and 5 μL
TEMED. The last two components should be added just
before use.
5. Mini-PROTEAN
® Electrophoresis Equipment (Bio-Rad) (see
Note 1).
6. Thermoblock.
7. Spin centrifuge.
2.3 Gel Staining
and Fractionation
1. Oriole™ Fluorescent Gel Stain (BioRad).
2. 70% Ethanol or methanol (for cleaning).
3. Square disposable Petri dishes.
4. Aluminum foil (to cover the container during staining).
Shotgun Proteomics in Non-model Organisms
81
5. Protein quantification reagents/kit.
6. Protein low bind microtubes.
2.2 1-D SDS-PAGE
1. Loading buffer: 180 mM Tris–HCl (pH 6.8), 6% SDS, 15%
β-mercaptoethanol, 30% glycerol, 0.15% bromophenol blue.
Prepare 1 M stock of Tris–HCl (pH 6.8) dissolving 12.114 g
Tris in 100 mL ultrapure water and fix the pH with few drops of
HCl 1 M. Prepare 10 mL of loading buffer adding 1.8 mL of
1 M Tris–HCl, 0.6 g SDS, 1.5 mL of β-mercaptoethanol, 3 mL
of glycerol, and 0.015 g of bromophenol blue in ultrapure
water. Store at À20
C in 1 mL aliquots.
2. Running buffer: 25 mM Tris, 0.19 M glycine, 0.1% SDS. Prepare 10% SDS by dissolving 10 g in 100 mL of ultrapure water.
Prepare a 5Â buffer by dissolving 15.175 g Tris, 71.4 g glycine,
and 50 mL 10% SDS in 1 L of distilled water. Dilute 1/5 before
using.
3. Running gel: 12.5% polyacrylamide gel. Prepare 100 mL of
1.5 M Tris–HCl, pH 8.8, dissolving 18.17 g of Tris. Prepare
10% ammonium persulfate (APS), dissolving 10 g APS in
100 mL. Mix all components in the following order, stirring
after each addition: 3.175 mL of ultrapure water, 4.17 mL of
30% acrylamide/bisacrylamide solution (ratio 37.5:1), 2.5 mL
of 1.5 M Tris–HCl (pH 8.8), 100 μL of 10% SDS, 50 μL of 10%
APS (freshly prepared), and 5 μL TEMED. The last two components should be added just before using.
4. Stacking gel: 4% acrylamide gel. Prepare 0.5 M Tris–HCl
(pH 6.8) by dissolving 6.057 g Tris in 100 mL ultrapure
water and fix the pH with a few drops of HCl 1 M. Mix all
components in the following order, stirring after each addition:
3 mL of ultrapure water, 660 μL of 30% acrylamide/bisacrylamide solution (ratio 37.5:1), 1.26 mL of 0.5 M Tris–HCl
(pH 6.8), 50 μL of 10% SDS, 25 μL of 10% APS, and 5 μL
TEMED. The last two components should be added just
before use.
5. Mini-PROTEAN
® Electrophoresis Equipment (Bio-Rad) (see
Note 1).
6. Thermoblock.
7. Spin centrifuge.
2.3 Gel Staining
and Fractionation
1. Oriole™ Fluorescent Gel Stain (BioRad).
2. 70% Ethanol or methanol (for cleaning).
3. Square disposable Petri dishes.
4. Aluminum foil (to cover the container during staining).
Shotgun Proteomics in Non-model Organisms
81