Loading of NTA and IDA agarose with metal ions
This protocol describes the loading of PureCube NTA or IDA Agarose with transition metal solutions, to obtain Co-/Cu-/Al-/Fe-/Zn-NTA or IDA. If you want to load nickel onto your agarose beads: Please refer to the appropriate protocol as nickel requires special care for optimal loading.
Amounts given in this protocol are for 20 mL NTA or IDA of 50% Agarose suspension, which contains 10 mL agarose. The reaction can be linearly scaled up and down as required.
Amounts given in this protocol are for 20 mL NTA or IDA of 50% Agarose suspension, which contains 10 mL agarose. The reaction can be linearly scaled up and down as required.
Composition of Buffers for metal ion loading
Sodium acetate buffer, pH 6.0, 100 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for buffer |
---|---|---|---|---|
Sodium acetate trihydrate | 50 mM | 136.08 | n.a. | 680 mg |
Instructions: Dissolve sodium acetate in 80 mL water, adjust the pH to 6.0 with acetic acid. Add water to a total volume of 100 mL. |
Al(III)chloride
Co(II)chloride
Cu(II)chloride
Zn(II)chloride solution, 50 mL
Component | Final concentration | Amount needed for buffer |
---|---|---|
Al(III)chloride Co(II)chloride Cu(II)chloride Zn(II)chloride | 2.5% (w/v) | 1.25 g |
Instructions: Dissolve in 50 ml water. |
Fe(II)chloride solution, 50 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for buffer |
---|---|---|---|---|
Fe(II)chloride hexahydrate | 2.5% (w/v) | 1.25 g | ||
Hydrochloric acid | 20 mM | 36.46 (density: 1.2 g/mL) | 1 M (ca.3%) | 1 mL |
Instructions: Dissolve iron (II) chloride in 40 mL water, then add hydrochloric acid. Add water to a total volume of 50 mL. |
Tris buffer, pH 7.5, 200 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for buffer |
---|---|---|---|---|
Tris base | 20 mM | 121.14 | 484 mg | |
Instructions: Dissolve Tris base in 160 mL water, adjust the pH to 7.5 with hydrochloric acid. Add water to a total volume of 200 mL. |
Agarose Storage Buffer, pH 6.5, 50 mL
Component | Final concentration | Molecular weight (g/mol) | Stock concentration | Amount needed for buffer |
---|---|---|---|---|
Sodium acetate trihydrate | 20 mM | 136.08 | n.a. | 135 mg |
Ethanol | 20 % (v/v) | 100 % (v/v) | 10.2 mL | |
Instructions: Dissolve sodium acetate in 30 mL water, adjust the pH to 6.5 with acetic acid. Add 9.6 mL water and 10.2 mL ethanol to yield a total volume of 50 mL. |
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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