Getting Started
You have a sample of biomolecules (e.g., protein, like T7 RNAP) that is (1) in the wrong buffer (e.g., has 500 mM Imidazole) and (2) is too dilute. Here’s what you can do about it.
Materials and Equipment
Prerequisite Protocols
Prepare P1 buffer: 
Make Protein Purification Buffers.
Make Protein Purification Buffers.Purchase (or assemble) buffer exchange columns: 
Prepare Columns.
Prepare Columns.Prepare protein samples: 
Perform Ni-His Purification by Gravity Column.
Perform Ni-His Purification by Gravity Column.(optionally) measure protein concentrations: 
BCA Assay and 
Protein Gel.
BCA Assay and Protein Gel.
Protocol
Equilibrate buffer exchange columns.
Load four (4) column volumes (4x ~ 3.5 mL) of cold P1 on top of buffer exchange columns.
Allow buffer to flow through.
Load 2.5 mL of sample onto each column. Allow buffer to flow through.
‣
Elute sample.
Load 3.5 mL cold P1 buffer onto column.
Capture flow through in a 15 mL centrifuge tube.
‣
Concentrate samples by spin filtration.
Load 500 uL of each protein sample onto Vivaspin 5 kDa cutoff spin columns.
Centrifuge samples at 12 000 rcf / 20 min (preferably cold) OR until sample is sufficiently concentrated (we concentrated to 25 uL each).
Resources and References
Other Protocols
Papers
Resources
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
Developers
Testers