Brief Description:
Reagent is good for washing beads after affinity isolation of the target protein.
Recipe:
STEN buffers (Salt, Tris, EDTA, NP40) are good for washing beads after affinity isolation of the target protein. Washing stringency can be increase by adding additional NaCl or SDS.
Stock Solutions
(1) 2 M Tris HCl, pH 6.8 (Trizma Hydrochloride, 250 mL)
(2 mol/L)(0.25L)(157.6g/mol) = 78.8g
- pH to 6.8: Add 13 mL 10 M NaOH, adjust accordingly
(2) 2.5 M NaCl (250 mL)
(2.5 mol/L)(0.25L)(58.44g/mol) = 36.525
STEN Buffer
| Stock |
Volume |
Final |
|
2 M Tris, pH 7.6
|
25 mL |
50 mM
|
|
2.5 M NaCl
|
60 mL
|
150 mM
|
|
0.5 M EDTA, pH 8.0
|
4 mL
|
2 mM
|
|
100% NP-40
|
2 mL
|
0.2%
|
|
Saturated PMSF (20mM in isopropanol)
|
2.5 mL
|
50 μM
|
|
Leupeptin (5mg/ml in H20)
|
1 mL
|
5 μg/ml
|
|
H20
|
905.5 mL
|
|
| |
1000 mL
|
|
0.5 M NaCl STEN
| Stock |
Volume |
Final |
|
2 M Tris, pH 7.6
|
25 mL |
50 mM
|
|
2.5 M NaCl
|
200 mL
|
500 mM
|
|
0.5 M EDTA, pH 8.0
|
4 mL
|
2 mM
|
|
100% NP-40
|
2 mL
|
0.2%
|
|
Saturated PMSF (20mM in isopropanol)
|
2.5 mL
|
50 μM
|
|
Leupeptin (5mg/ml in H20)
|
1 mL
|
5 μg/ml
|
|
H20
|
765.5 mL
|
|
| |
1000 mL
|
|
SDS STEN
| Stock |
Volume |
Final |
|
2 M Tris, pH 7.6
|
25 mL |
50 mM
|
|
2.5 M NaCl
|
60 mL
|
150 mM
|
|
0.5 M EDTA, pH 8.0
|
4 mL
|
2 mM
|
|
100% NP-40
|
2 mL
|
0.2%
|
|
Saturated PMSF (20mM in isopropanol)
|
2.5 mL
|
50 μM
|
|
Leupeptin (5mg/ml in H20)
|
1 mL
|
5 μg/ml
|
|
H20
|
900.5 mL
|
|
|
20% SDS
|
5 mL |
0.1% |
| |
1000 mL |
|