Washing and Regenerating His Affinity MagBeads and Agarose Resin
NTA and IDA agarose resins and MagBeads should be washed after each run, and regenerated latest after 5 runs (though we recommend to regenerate the resin after each run, if possible). This protocol delineates washing and regenerating procedures for our His Affinity products, including a specific procedure for resins that have been exposed to reducing agents such as DTT. 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. This protocol can also be implemented for NTA and IDA resins loaded with other metals (e.g., Co, Fe, Al, Cu), using the appropriate solutions to recharge the resin. The novel INDIGO ligand cannot be stripped with EDTA.
Buffer compositions - His-tag regeneration protocol.
Wash Buffer, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
---|---|---|---|---|---|
NaOH | 0.5 M | 39.997 | 1 M | 4 g/ 100 mL | 50 mL |
NaCl | 2.0 M | 58.440 | 5 M | 29.2 g/ 100 mL | 40 mL |
LDAO* | 2% (v/v) | 229.40 | 30% (w/v) | 0.3 g/ 1 mL | 6.6 mL |
Instructions: Mix components. Add dd water to a final volume of 100 mL. Additionally, PureCube NTA, PureCube 100 NTA and PureCube IDA are compatible with the following cleaning reagents: 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile, 1 M NaOH. * LDAO is only required if a membrane protein was purified on the resin being washed or regenerated. An alternate detergent may be used but generally we recommend LDAO because it is non-ionic, harsh to proteins, and easily washed off the resin. |
100 mM EDTA, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Amount needed |
---|---|---|---|
EDTA | 100 mM | 292.24 | 2.922 g/ 100 mL |
Instructions: Weigh EDTA, fill up to 100 mL dd water and mix well. |
10 mM NiSO4 , 100 mL
Component | Final concentration | Molecular weight (g/mol) | Amount needed |
---|---|---|---|
NiSO4 | 10 mM | 154.75 | 0.155 g/ 100 mL |
Instructions: Weigh NiSO4, fill up to 100 mL dd water and mix well. |
Regeneration Buffer, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for stock | Stock needed for buffer |
---|---|---|---|---|---|
Acetic acid (97%) | 100 mM | 60.05 | 10%=0.17 M | 10.3 mL/100 mL | 6.05 mL |
NaCl | 150 mM/td> | 58.44 | 5 M | 29.2 g/ 100 mL | 1.5 mL |
Instructions: Add components to 80 mL dd water. Mix well and fill up to 100 mL.. |
20 mM Tris, pH 8.0, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Amount needed |
---|---|---|---|
Tris base | 20 mM | 121.14 | 242 mg |
Instructions: Dissolve in 80 mL dd water. Set pH to 8.0 using HCl and fill up to 100 mL. |
A. | Wash protocol - Recommended after each use |
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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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B. | Wash & Regeneration protocol without the use of DTT (recommended after each run, latest after 5 runs) |
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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C. | Wash & Regeneration protocol after the use of DTT (Neccessary after each run!) |
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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