Getting Started
You have a sample of a biomolecule (e.g., T7RNAP, tRNA, ribosomes, plasmid, etc.) that is either (1) in the wrong buffer (e.g., has 500 mM Imidazole) or (2) is too dilute.
You can use centrifugal spin filters to concentrate your sample and change its buffer. These filters allow buffer and small molecules to flow through them, retaining large molecules bigger than the filter’s specified molecular weight.
Materials and Equipment
We use two different volume spin filters, depending on how much sample volume we need to process: 15 mL or 0.5 mL.
Name | Product | Manufacturer | Part # | Price | Storage Conditions | Link |
15 mL spin filters (3 kDa cutoff) | Amicon® Ultra Centrifugal Filter, 3 kDa MWCO | Millipore Sigma | UFC9003 | $125 | 4C to 30C | [link] |
0.5 mL spin filters (3 kDa cutoff) | Amicon® Ultra Centrifugal Filter, 3 kDa MWCO | Millipore Sigma | UFC5003 | $63 | 4C to 30C | [link] |
Protocol
(If exchanging buffers) dilute sample ~ 10x with the final buffer (e.g., 5 mL protein in P2 elution buffer + 45 mL P3 Exchange Buffer).
Load sample onto the spin column.
Centrifuge samples at 4000 rcf for 10 min at 4C.
Repeat loading and spinning until all the sample has been processed.
(If exchanging buffers) dilute sample ≥ 10x further with the final buffer and repeat loading and spinning until all the sample has been processed.
Resources and References
- Papers
- Original PURE paper
- OnePot PURE
Credits
Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)
- Developers