Batch Spin His-tag Purification using PureCube Resins

This protocol applies to all agarose resins that are listed as His affinity Products in our online shop!

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This protocol describes how to purify His-tagged using batch spin with our His-affinity purification resins. The recipes for the mentioned buffer are given below. Alternatively the HisCube buffers can also be purchased directly.

The protocol given here is a referring to protein purification under NATIVE conditions. For the denaturing conditions please refer to the PDF files above.

Watch this video to see the protocol in action.

Buffer compositions

Bind Buffer, 50 mL

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

NaH₂PO₄	50 mM	119.98	0.5 M	29.99 g/ 500 mL	5 mL
NaCl	300 mM	58.44	5 M	146.1 g/ 500 mL	3 mL
Imidazole	10 mM	68.08	1 M	6.8 g/ 100 mL	0.5 mL
Instructions: Mix in 40 mL water. Adjust the pH to 8.0 using NaOH and then add water to a total volume of 50 mL. Always prepare fresh.

Wash Buffer, 100 mL

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

NaH₂PO₄	50 mM	119.98	0.5 M	29.99 g/ 500 mL	10 mL
NaCl	300 mM	58.44	5 M	146.1 g/ 500 mL	6 mL
Imidazole	20 mM	68.08	1 M	6.8 g/ 100 mL	2 mL
Instructions: Mix in 80 mL water. Adjust the pH to 8.0 using NaOH and then add water to a total volume of 100 mL. Always prepare fresh.

Elution Buffer, 50 mL

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

NaH₂PO₄	50 mM	119.98	0.5 M	29.99 g/ 500 mL	5 mL
NaCl	300 mM	58.44	5 M	146.1 g/ 500 mL	3 mL
Imidazole*	500 mM	68.08	1 M	6.8 g/ 100 mL	25 mL
Instructions: Mix in 40 mL water. Adjust the pH to 8.0 using NaOH and then add water to a total volume of 50 mL. Always prepare fresh.

* Tag length and protein structure can impact the interaction between His-tag and nickel ion. Therefore, we recommend trying a concentration gradient of imidazole to find the minimum concentration that elutes the desired amount of protein from the column.

Protocol


A.	Protocol for His-tag batch spin purification under native conditions.:
	Pipette 1 ml Ni-INDIGO agarose slurry into a reaction tube. Centrifuge the agarose particles at 500 x g. Then remove the storage buffer on top. Add binding buffer to the agarose. The recipes for all required buffers can be found in the description below. Shake the reaction tube. Remove excess binding buffer by centrifuging at 500 xG. We recommend to repeat the two previous steps (3 and 4 once). Now add the His-tagged protein lysate to the reaction tube. Let the mixture incubate in the fridge shaking for one hour at 4°C. Centrifuge the mixture at 500 x g. Remove the excess protein lysate. Add wash buffer. Let the mixture incubate in the fridge shaking for one hour at 4°C. Centrifuge the mixture at 500 x g. Then remove the wash buffer on top. Repeat the washing procedure (Steps 10-12) for 4 times. Add 1 ml Elution Buffer. Let the mixture incubate in the fridge shaking at least 10 minutes at 4°C. Centrifuge the elution buffer and the agarose at 500 x g. We recommend to repeat this and the two previous steps at least twice. Pipette the elution buffer on top into a new reaction tube. The new reaction tube now contains your purified His-tagged protein.

Protocol for His-tag batch spin purification under native conditions.:

Pipette 1 ml Ni-INDIGO agarose slurry into a reaction tube.
Centrifuge the agarose particles at 500 x g. Then remove the storage buffer on top.
Add binding buffer to the agarose. The recipes for all required buffers can be found in the description below.
Shake the reaction tube.
Remove excess binding buffer by centrifuging at 500 xG. We recommend to repeat the two previous steps (3 and 4 once).
Now add the His-tagged protein lysate to the reaction tube.
Let the mixture incubate in the fridge shaking for one hour at 4°C.
Centrifuge the mixture at 500 x g.
Remove the excess protein lysate.
Add wash buffer.
Let the mixture incubate in the fridge shaking for one hour at 4°C.
Centrifuge the mixture at 500 x g. Then remove the wash buffer on top.
Repeat the washing procedure (Steps 10-12) for 4 times.
Add 1 ml Elution Buffer.
Let the mixture incubate in the fridge shaking at least 10 minutes at 4°C.
Centrifuge the elution buffer and the agarose at 500 x g. We recommend to repeat this and the two previous steps at least twice.
Pipette the elution buffer on top into a new reaction tube.
The new reaction tube now contains your purified His-tagged protein.