21. Soluble fraction buffer: 10 mM Tris–HCl, pH 8.0,
150 mM NaCl.
22. Insoluble fraction buffer: 10 mM Tris–HCl, pH 8.0, 150 mM
NaCl, 8 M urea.
23. Urea: 9 M solution in water. Dissolve 540.54 g urea in 600 mL
of warm water (~30
C), and mix vigorously with a stirring bar.
Afterward, add water to a final volume of 1 L.
24. Imidazole: 2 M solution in water.
25. Binding buffer: 100 mM Tris–HCl, pH 8.0, 500 mM NaCl,
5 mM imidazole.
26. Wash buffer: 100 mM Tris–HCl, pH 8.0, 500 mM NaCl,
20–100 mM imidazole.
27. Elution buffer: 100 mM Tris–HCl, pH 8.0, 500 mM NaCl,
1 M imidazole.
28. 1.5 mL microtubes.
29. Chromatographic column.
30. Chelating Sepharose.
31. Nickel sulfate (Ni
2+
): 0.3 M solution in water.
32. Thirty percent acrylamide/BIS solution (29:1) acrylamide:
BIS: Weigh out 29 g acrylamide and 1 g bisacrylamide and
add water to a final volume of 100 mL, with shaking until
totally dissolved. Filter the solution through a 0.22 μm filter,
and store at 4
C, in a bottle wrapped with aluminum foil.
33. Ammonium persulfate: 10% solution in water.
34. N,N,N
0 ,N
0 -Tetramethylethylenediamine (TEMED): store at
4
C.
35. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Autoclave and
store at room temperature.
36. Stacking gel buffer: 1 M Tris–HCl, pH 6.8. Autoclave and
store at room temperature.
37. SDS-PAGE sample buffer (5Â): 250 mM Tris–HCl, pH 6.8,
0.1% SDS, 50% glycerol, 6.7% β-mercaptoethanol, 0.5 mg/mL
bromophenol blue. Mix 7.5 mL of 1 M Tris–HCl, pH 6.8,
0. 3 mL of 10% SDS, 15 mL of glycerol, and 15 mg bromophenol blue in water a final volume of 28 mL, and heat the
solution at 65
C for total dissolution. At the end, add 2 mL of
β-mercaptoethanol.
38. Sodium dodecyl sulfate (SDS): 10% solution in water.
39. Tris-glycine buffer (5Â): 25 mM Tris–HCl, pH 8.3, 192 mM
glycine, 0.5% SDS. Store at room temperature.
40. Glass plates.
41. Gel caster.
174
Aline F. Teixeira and Ana L. T. O. Nascimento
150 mM NaCl.
22. Insoluble fraction buffer: 10 mM Tris–HCl, pH 8.0, 150 mM
NaCl, 8 M urea.
23. Urea: 9 M solution in water. Dissolve 540.54 g urea in 600 mL
of warm water (~30
C), and mix vigorously with a stirring bar.
Afterward, add water to a final volume of 1 L.
24. Imidazole: 2 M solution in water.
25. Binding buffer: 100 mM Tris–HCl, pH 8.0, 500 mM NaCl,
5 mM imidazole.
26. Wash buffer: 100 mM Tris–HCl, pH 8.0, 500 mM NaCl,
20–100 mM imidazole.
27. Elution buffer: 100 mM Tris–HCl, pH 8.0, 500 mM NaCl,
1 M imidazole.
28. 1.5 mL microtubes.
29. Chromatographic column.
30. Chelating Sepharose.
31. Nickel sulfate (Ni
2+
): 0.3 M solution in water.
32. Thirty percent acrylamide/BIS solution (29:1) acrylamide:
BIS: Weigh out 29 g acrylamide and 1 g bisacrylamide and
add water to a final volume of 100 mL, with shaking until
totally dissolved. Filter the solution through a 0.22 μm filter,
and store at 4
C, in a bottle wrapped with aluminum foil.
33. Ammonium persulfate: 10% solution in water.
34. N,N,N
0 ,N
0 -Tetramethylethylenediamine (TEMED): store at
4
C.
35. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Autoclave and
store at room temperature.
36. Stacking gel buffer: 1 M Tris–HCl, pH 6.8. Autoclave and
store at room temperature.
37. SDS-PAGE sample buffer (5Â): 250 mM Tris–HCl, pH 6.8,
0.1% SDS, 50% glycerol, 6.7% β-mercaptoethanol, 0.5 mg/mL
bromophenol blue. Mix 7.5 mL of 1 M Tris–HCl, pH 6.8,
0. 3 mL of 10% SDS, 15 mL of glycerol, and 15 mg bromophenol blue in water a final volume of 28 mL, and heat the
solution at 65
C for total dissolution. At the end, add 2 mL of
β-mercaptoethanol.
38. Sodium dodecyl sulfate (SDS): 10% solution in water.
39. Tris-glycine buffer (5Â): 25 mM Tris–HCl, pH 8.3, 192 mM
glycine, 0.5% SDS. Store at room temperature.
40. Glass plates.
41. Gel caster.
174
Aline F. Teixeira and Ana L. T. O. Nascimento