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!
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.: |
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