Protein Protocols Protein Protocols

74 Judd
2. Materials
2.1. Equipment
1. SDS-PAGE gel apparatus.
2. Power pack.
3. Blotting apparatus.
2.2. Reagents
1. Separating/spacer gel acrylamide (1X crosslinker): 48 g acrylamide, 1.5 g N,N'-methylene-bis-acrylamide.
Bring to 100 mL, and then filter through qualitative paper to remove
cloudiness (see Note 1).
2. Separating gel acrylamide (2X crosslinker): 48 g acrylamide, 3 g N,N'-methylenebis-acrylamide.
Bring to 100 mL, and then filter through qualitative paper to remove cloudiness
(see Note 2).
3. Stacking gel acrylamide: 30 g acrylamide, 0.8 g N,N'-methylene-bis-acrylamide. Bring to
100 mL, and then filter through qualitative paper to remove cloudiness.
4. Separating/spacer gel buffer: 3 M Trizma base, 0.3% sodium dodecyl sulfate (see Note 3).
Bring to pH 8.9 with HCl.
5. Stacking gel buffer: 1 M Tris-HCl, pH 6.8.
6. Cathode (top) running buffer (10X stock): 1 M Trizma base, 1 M tricine, 1% SDS (see
Note 3). Dilute 1:10 immediately before use. Do not adjust pH; it will be about 8.25.
7. Anode (bottom) buffer (10X stock): 2 M Trizma base. Bring to pH 8.9 with HCl. Dilute
1:10 immediately before use.
8. 0.2 M tetrasodium EDTA.
9. 10% ammonium persulfate (make fresh as required).
10. TEMED.
11. Glycerol.
12. Fixer/destainer: 25% isopropanol, 7% glacial acetic acid in dH2O (v/v/v).
13. 1% Coomassie brilliant blue (CBB) (w/v) in fixer/destainer.
14. Sample solubilization buffer: 2 mL 10% SDS (w/v) in dH2O, 1.0 mL glycerol, 0.625 mL
1 M Tris-HCl, pH 6.8, 6 mL dH2O, bromphenol blue to color.
15. Dithiothreitol.
16. 2% Agarose.
17. Molecular-mass markers, e.g., low-mol-wt kit (Bio-Rad, Hercules, CA), or equivalent, and
peptide molecular-mass markers (Pharmacia Inc., Piscataway, NJ), or equivalent.
18. PVDF (nylon) membranes.
19. Methanol.
20. Blotting transfer buffer: 20 mM phosphate buffer, pH 8.0: 94.7 mL 0.2 M Na2HPO4 stock,
5.3 mL 0.2 M NaH2PO4 stock in 900 mL H2O. 21. Filter paper for blotting (Whatman No. 1), or equivalent.
22. Distilled water (dH2O). 2.3. Gel Recipes
2.3.1. Separating Gel Recipe
Add reagents in order given (see Note 4): 6.7 mL water, 10 mL separating/spacer gel
buffer, 10 mL separating/spacer gel acrylamide (1X or 2X crosslinker), 3.2 mL glycerol,
10 µL TEMED, 100 µL 10% ammonium persulfate.