Encyclopedia of Experiments: Biological Techniques
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Transcript
Begin this protocol with inducible over-expression of a histidine-tagged protein as described in the text protocol. Prepare 1 milliliter of Nickel-Nitriloacetic Acid resin in a gravity column, to purify the protein. The day before use, equilibrate the column overnight at 4 degrees Celsius with 2 milliliters of equilibration buffer.
The following day, bring the column from 4 degrees Celsius to room temperature, prior to loading the clarified lysate, and let it stand for approximately two to three hours. Then, re-suspend the pellet in the lysis buffer.
Sonicate the cells on ice for 10 times 10-second intervals, pausing 30 seconds between pulses. Clarify the lysate by centrifugation at 3080 x g for 30 minutes at 4 degrees Celsius using a microcentrifuge.
Prepare the clarified lysate with equal volumes of lysis buffer, then, apply the prepped clarified lysate to the column, and collect the flow-through. Re-apply the clarified lysate flow-through to the column, and collect the secondary flow-through.
Then, wash the column with 5 milliliters of wash buffer #1 and collect the flow-through. Wash the column again with 5 milliliters of wash buffer #2, and collect the flow-through. Now, apply 2 milliliters of elution buffer. Collect flow-through in two fractions of 1 milliliter each.
