Washing and Regenerating of Strep Agarose
HiCap StrepTactin Agarose can be regenerated for re-use according to the protocol delineated below. Desthiobiotin is removed by an incubation with 2-(4-Hydroxyphenylazo)benzoic acid (HABA), followed by wash steps. Volumes are given in column bed volume (bv), i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume. The material can be regenerated 3-5 times. Note that a regeneration is only possible if desthiobiotin has been used for elution. If biotin has been used, binding to the agarose matrix is irreversible
Composition of Buffers for Strep Agarose Regeneration
Regeneration Buffer, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
---|---|---|---|---|---|
TRIS base, pH 8.0 | 100 mM | 121.14 | 1 M | 60.57 g/ 500 mL | 10 mL |
NaCl | 150 mM | 58.44 | 5 M | 146.1 g/500 mL | 3 mL |
HABA | 1 mM | 242.23 | - | 24 mg | - |
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0. Alternatively, prepare 200 mL Wash Buffer and separate in 2 x 100 mL, add HABA to one aliquot |
Wash Buffer, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
---|---|---|---|---|---|
TRIS base, pH 8.0 | 100 mM | 121.14 | 1 M | 60.57 g/ 500 mL | 10 mL |
NaCl | 150 mM | 58.44 | 5 M | 146.1 g/500 mL | 3 mL |
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0. |
Wash Buffer, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
---|---|---|---|---|---|
TRIS base, pH 8.0 | 100 mM | 121.14 | 1 M | 60.57 g/ 500 mL | 10 mL |
EDTA | 1.3 mM | 292.24 | 0.5 M | 14.6 g/100 mL | 260 µL |
Sodium azide | 0.02% | - | - | - | 20 mg |
Instructions: Prepare a TRIS base stock solution and set the pH with HCl to 8.0. |
A. | Procedure |
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Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
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