Washing and Regenerating His Affinity MagBeads and Agarose Resin

NTA and IDA agarose resins and MagBeads should be washed after each run, and regenerated latest after 5 runs (though we recommend to regenerate the resin after each run, if possible). This protocol delineates washing and regenerating procedures for our His Affinity products, including a specific procedure for resins that have been exposed to reducing agents such as DTT. Volumes are given in column bed volume (bv), i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume. This protocol can also be implemented for NTA and IDA resins loaded with other metals (e.g., Co, Fe, Al, Cu), using the appropriate solutions to recharge the resin. The novel INDIGO ligand cannot be stripped with EDTA.

Buffer compositions - His Tag purification with Midi and Mini 1-step batch columns.

Wash Buffer, 100 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

NaOH	0.5 M	39.997	1 M	4 g/ 100 mL	50 mL
NaCl	2.0 M	58.440	5 M	29.2 g/ 100 mL	40 mL
LDAO*	2% (v/v)	229.40	30% (w/v)	0.3 g/ 1 mL	6.6 mL
Instructions: Mix components. Add dd water to a final volume of 100 mL. Additionally, PureCube NTA, PureCube 100 NTA and PureCube IDA are compatible with the following cleaning reagents: 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% (v/v) acetonitrile, 1 M NaOH. * LDAO is only required if a membrane protein was purified on the resin being washed or regenerated. An alternate detergent may be used but generally we recommend LDAO because it is non-ionic, harsh to proteins, and easily washed off the resin.

100 mM EDTA, 100 mL

Component	Final concentration	Molecular weight (g/mol)	Amount needed

EDTA	100 mM	292.24	2.922 g/ 100 mL
Instructions: Weigh EDTA, fill up to 100 mL dd water and mix well.

10 mM NiSO₄ , 100 mL

Component	Final concentration	Molecular weight (g/mol)	Amount needed

NiSO₄	10 mM	154.75	0.155 g/ 100 mL
Instructions: Weigh NiSO₄, fill up to 100 mL dd water and mix well.

Regeneration Buffer, 100 mL

Component	Final concentration	Molecular weight (g/mol)	Stock concentration	Amount needed for stock	Stock needed for buffer

Acetic acid (97%)	100 mM	60.05	10%=0.17 M	10.3 mL/100 mL	6.05 mL
NaCl	150 mM/td>	58.44	5 M	29.2 g/ 100 mL	1.5 mL
Instructions: Add components to 80 mL dd water. Mix well and fill up to 100 mL..

20 mM Tris, pH 8.0, 100 mL

Component	Final concentration	Molecular weight (g/mol)	Amount needed

Tris base	20 mM	121.14	242 mg
Instructions: Dissolve in 80 mL dd water. Set pH to 8.0 using HCl and fill up to 100 mL.

A.	Wash protocol - Recommended after each use

	Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume. Remove the majority of the fluid in the column containing the the His affinity matrix. Add 10 bv dd water and allow the majority of the water volume to drip out of the column. Note: You can allow the fluid to drip through the column by gravity, or use a pressure bulb to gently force the fluid through the matrix. Ensure not to dry out the matrix. . Add 10 bv Wash Buffer to the column and allow the volume to completely flow through the matrix. Rinse the column again with 10 bv dd water. Add 10 bv 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
B.	Wash & Regeneration protocol without the use of DTT (recommended after each run, latest after 5 runs)
	Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume. Remove the majority of the fluid in the column containing the His Affinity matrix. Add 10 bv dd water and allow the majority of the volume to drip out of the column. Note: For removal of contaminations with very hydrophobic proteins or lipids, or precipitated proteins, incubate the matrix with one of the following chemicals for 1-2 h: 100% methanol, 100% ethanol, 8 M urea, 6 M guanidinium hydrochloride, 30% acetonitrile, or 1 M NaOH. Thoroughly wash with distilled water. Add 10 bv 100 mM EDTA to the column and allow the entire volume to flow through the matrix. Rinse the column again with 10 bv dd water Add 10 bv Wash Buffer to the column and allow the entire volume to flow through the matrix. Rinse the column with 10 bv dd water Add 10 bv 10m M NiSO₄ (depends on your resin!) to recharge the matrix. Allow the volume to drip through the column by gravity Rinse the column with 5 bv dd water. Add 5 bv of Regeneration Buffer and incubate the column for 15 min at room temperature. Wash twice with 5 bv dd water each Wash with 5 bv 20 mM Tris pH 8.0. Add 10 bv of 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.
C.	Wash & Regeneration protocol after the use of DTT (Neccessary after each run!)
	Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume. Remove the majority of the fluid in the column containing the His Affinity resin or MagBeads. Add 10 bv dd water and allow the majority of the volume to drip out of the column. Briefly wash the resin with 10 bv 1–3% (v/v) HCl. Minimize the exposure time of the resin to HCl. Note: The concentration of HCl depends on the extent to which the resin is reduced. For example, 1% HCl was sufficient to strip Ni-NTA MagBeads exposed to 1 mM DTT, 2% HCl for 5 mM DTT, and 3% for 10 mM DTT. Rinse the column with 10 bv dd water If the resin is not completely white, repeat steps 2 and 3. Otherwise, continue to step 5. Add 10 bv Wash Buffer and allow the majority of the volume to drip out of the column. Rinse the column with 10 bv dd water. Add 10 bv 10 mM NiSO₄ (depends on your resin!) to recharge the resin. Allow the volume to drip through the column by gravity. Rinse the column with 5 bv dd water Add 5 bv of Regeneration Buffer and incubate the matrix for 15 min at room temperature. Wash twice with 5 bv dd water each. Wash with 5 bv 20 mM Tris pH 8.0. Note: The extensive wash steps remove free nickel ions from the column, enhancing performance of the material in subsequent purifications, especially in presence of DTT. Wash twice with 5 bv dd water each . Add 10 bv 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. The matrix is now ready to be re-used.