6-His Fusion Protein Purification

Buffers for 6-His Fusion Protein Purification
0.5M phosphate, pH 8.0 500 ml
Na-phosphate dibasic (141.96) 35.5 g
pH with phosphoric acid
0.5 M phosphate, pH 6.0 250 ml
Na-phosphate dibasic (141.96) 8.88 g
Na-phosphate monobasic (119.96) 7.5 g
pH with phosphoric acid
Lysis Buffer (50 mM Pi, pH 8.0; 300 mM NaCl; 2M Urea; 0.1 % Tween 20; 10 mM Imidazole + 1 mM PMSF+
1 μg/ml PI cocktail + 1 mg/ml lysozyme)
100 ml
0.5 M phosphate, pH 8.0 10 ml
NaCl (58.44) 1.75 g
Urea (60.06) 12.01 g
Tween 20 100 μl
Imidazole (68.08) 68 mg
Column Buffer (50 mM Pi, pH 8.0; 500 mM NaCl; 0.1 % Tween 20; 10 mM Imidazole; 5 mM B-ME + 0.1 mM
PMSF+ 0.1 μg/ml PI cocktail + 0.04 % Na-azide)
500 ml
0.5 M phosphate, pH 8.0 50 ml
NaCl (58.44) 14.61 g
Tween 20 500 μl
Imidazole (68.08) 0.34 g
B-ME 175 μl
Column Equillibration Buffer
Column Buffer + 2M Urea. 6g urea/ 50 ml buffer
pH 6.0 Wash Buffer (50 mM Pi, pH 6.0; 500 mM NaCl; 5 mM B-ME + 0.1 mM PMSF+ 0.1 μg/ml PI cocktail +
0.04 % Na-azide)
50 ml
0.5 M phosphate, pH 6.0 5 ml
NaCl (58.44) 1.46 g
B-ME 17.5 μl
Elution Buffer (50 mM Pi, pH 7.2; 500 mM NaCl; 5 mM B-ME; 250 mM Imidazole + 0.04 % Na-azide)
50 ml
0.5 M phosphate, pH 7.2 5 ml
NaCl (58.44) 1.46 g
B-ME 17.5 μl
Imidazole (68.08) 0.85 g
Readjust the pH because the imidazole affects it.

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