Loading of PureCube NTA or IDA MagBeads with nickel

 

This protocol describes the loading of PureCube NTA or IDA MagBeads with nickel solutions, to obtain Ni-NTA or Ni-IDA MagBeads. Please refer to the appropriate protocol for loading with other transition metals. T Amounts given in this protocol are for 1 mL NTA or IDA MagBead suspension, which contains 250 µL magnetic beads. The Cube MagBead Separator holds 1.5 and 2 ml microfuge tubes for convenient separation of MagBeads from the supernatant. This reaction can be linearly scaled up or down. Magnetic holders for a wide range of volumes are available e.g. our own MagBead separator or the sepmag.eu .

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, 20 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for buffer
Nickel II sulfate hexahydrate 2.5% (w/v) 262.85 n.a. 500 mg
Instructions: Dissolve in 20 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, 100 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for buffer
Tris base 20 mM 121.14   242 mg
Instructions: Dissolve Tris base in 80 mL water, adjust the pH to 7.5 with hydrochloric acid. Add water to a total volume of 100 mL.

MagBead Storage Buffer, pH 6.5, 250 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 1 mL PureCube NTA or IDA MagBeads into a 2 mL microcentrifuge tube.
  2. Place the tube on a magnetic stand and allow the beads to separate. Remove the supernatant. Resuspend the magnetic beads with 1 mL double distilled water.
  3. Separate the beads and wash two more times with water.
  4. Wash 3x with 50 mM sodium acetate, pH 6.0.
  5. Wash 1x with double distilled water.
  6. Add 1 ml 2.5% nickel sulfate solution and incubate for 2 h on an end-over-end shaker.
  7. Wash 4x with double distilled water.
  8. Add 1 mL Wash Buffer and incubate for 10 min.
  9. Wash 1x with double distilled water.
  10. Wash 6x with 20 mM Tris-HCl, pH 7.5.
  11. Wash 1x with double distilled water.
  12. Resuspend the Ni-NTA or Ni-IDA MagBeads in 1 mL MagBead Storage buffer, yielding a 25% suspension. Store at 4°C.
  Tip: The loading reaction can be scaled up and down linearly, by increasing or decreasing the amounts of buffers and solutions described in this protocol.
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