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

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)	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
	Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume. Transfer 20 mL PureCube NTA or IDA Agarose suspension into a 50 mL centrifuge tube. Spin the tube for 5 min at 500 x g to pellet the agarose. Remove the supernatant. Resuspend with 20 mL double distilled water. Wash two more times with 20 mL water. Wash 3x with 20 mL 50 mM sodium acetate, pH 6.0. Wash 1x with 20 mL double distilled water. 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. Wash 4x with 20 mL double distilled water. Wash 6x with 20 mL 20 mM Tris-HCl, pH 7.5 Wash 1x with 20 mL double distilled water. Resuspend the Ni-NTA or Ni-IDA Agarose in 20 mL Agarose Storage buffer, yielding a 50% suspension. Store at 4°C.

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.

Transfer 20 mL PureCube NTA or IDA Agarose suspension into a 50 mL centrifuge tube.
Spin the tube for 5 min at 500 x g to pellet the agarose. Remove the supernatant. Resuspend with 20 mL double distilled water.
Wash two more times with 20 mL water.
Wash 3x with 20 mL 50 mM sodium acetate, pH 6.0.
Wash 1x with 20 mL double distilled water.
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.
Wash 4x with 20 mL double distilled water.
Wash 6x with 20 mL 20 mM Tris-HCl, pH 7.5
Wash 1x with 20 mL double distilled water.
Resuspend the Ni-NTA or Ni-IDA Agarose in 20 mL Agarose Storage buffer, yielding a 50% suspension. Store at 4°C.