Loading of PureCube NTA or IDA Agarose with nickel

 

This protocol describes the loading of PureCube NTA or IDA Agarose with nickel solutions, to obtain Ni-NTA or Ni-IDA Agarose. Please refer to the appropriate protocol for loading with other transition metals. 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 Nickel (Ni) 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.

Nickel sulfate solution, 50 mL

 
ComponentFinal concentrationAmount needed for buffer
Nickel II sulfate hexahydrate 2.5% (w/v) 1.25 g
Instructions: Dissolve in 50 ml water.

Wash Buffer, 100 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for buffer
Sodium chloride 150 mM 58.44 n.a. 877 mg
Acetic acid 200 mM 60.05 Density 1.05 g/mL   1.14 mL
Instructions: Dissolve sodium chloride in 80 mL water, then add acetic acid. Add water to a total volume of 100 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 sodium chloride in 80 mL water, then add acetic acid. Add water to a total volume of 100 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% nickel sulfate solution and incubate for 2 h.
  7. Wash 4x with 20 mL double distilled water.
  8. Add 20 mL Wash Buffer and incubate for 10 min.
  9. Wash 1x with 20 mL double distilled water.
  10. Wash 6x with 20 mL 20 mM Tris-HCl, pH 7.5.
  11. Wash 1x with 20 mL double distilled water
  12. 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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