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.

Composition of Buffers for metal ion loading

Sodium acetate buffer, pH 6.0, 100 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount 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

 
ComponentFinal concentrationAmount 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

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount 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

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount 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

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount 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
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. Transfer 20 mL PureCube NTA or IDA Agarose suspension into a 50 mL centrifuge tube.
  2. Spin the tube for 5 min at 500 x g to pellet the agarose. Remove the supernatant. Resuspend with 20 mL double distilled water.
  3. Wash two more times with 20 mL water.
  4. Wash 3x with 20 mL 50 mM sodium acetate, pH 6.0.
  5. Wash 1x with 20 mL double distilled water.
  6. Add 20 ml 2.5% transition metal solution and incubate for 2 h on an end-over-end shaker. Note: Ensure to add HCl when loading NTA or IDA Agarose with iron chloride.
  7. Wash 4x with 20 mL double distilled water.
  8. Wash 6x with 20 mL 20 mM Tris-HCl, pH 7.5
  9. Wash 1x with 20 mL double distilled water.
  10. Resuspend the Ni-NTA or Ni-IDA Agarose in 20 mL Agarose Storage buffer, yielding a 50% suspension. Store at 4°C.
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