Getting Started
You have a protein that you want to purify out of a complicated mixture. This protein has a 6xHis purification tag, which chelates Ni ions. You can use a Ni-His resin to bind thusly tagged proteins. This resin is a suspension of microscopic gel beads with Ni ions covalently linked. Bound proteins can then be washed to remove non-tagged proteins, then eluted in a buffer with high concentration (~ 500 mM) imidazole. Imidazole chelates the Ni ions, competing with and ultimately displacing the 6xHis tagged proteins.
Materials and Equipment
Prerequisite Protocols
Prepare P1, P2 and 2x Storage buffers: 
Make Protein Purification Buffers.
Make Protein Purification Buffers.Assemble and equilibrate columns: 
Prepare Columns.
Prepare Columns.Prepare clarified lysates: 
Prepare Bacterial Lysates.
Prepare Bacterial Lysates.(Optionally) measure protein concentrations: 
BCA Assay and 
Protein Gel.
BCA Assay and Protein Gel.(Optionally) exchange buffers and concentrate samples: 
Perform Buffer Exchange and Concentration.
Perform Buffer Exchange and Concentration.
Protocol
Per sample, prepare:
50 mL cold P1 buffer.
5 mL cold P2 buffer.
2 mL cold 2x storage buffer.
1x gravity column (1 mL resin bed).
Equilibrate column with ≥ 10 mL cold P1 buffer
Load up to 10 mL clarified lysate per column. sample to Allow flow through. (Optionally) capture for later analysis; see BCA Assay and Protein Gel.
BCA Assay and Protein Gel.‣
Wash column with at least 10 mL cold P1 buffer. Allow buffer to flow through. (Optionally) capture for later analysis; see BCA Assay and Protein Gel.
BCA Assay and Protein Gel.Elute sample in 2.5 mL cold P2 buffer. Capture flow through in 15 mL centrifuge tube.
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(Optionally) exchange buffers for each protein sample (see Perform Buffer Exchange and Concentration).
Perform Buffer Exchange and Concentration).(Optionally) concentrate each protein sample (see Perform Buffer Exchange and Concentration).
Perform Buffer Exchange and Concentration).(Optionally) prepare proteins for freezing.
Add 1x sample volume of 2x Storage buffer (e.g., 1 mL 2x Storage Buffer to 1 mL protein sample). Mix by pipetting.
Freeze proteins at -80C until ready for use.
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Resources and References
Other Protocols
Papers
Resources
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
Developers
Testers