Getting Started
In order to purify proteins, we first need a source of proteins to purify! We make proteins using bacterial strains that carry our protein of interest on an expression plasmid (here: pET28a). These expression plasmids put a gene of interest under the transcription of an inducible promoter (e.g., pT7). This allows us to first (1) grow our bacteria quickly, without the metabolic load of making a lot of proteins, before induction (so that we have a lot of cells to make proteins), then (2) to induce the expression of our protein of interest after induction (so that we have a lot of protein per cell, even if making so much protein is toxic to the cell).
Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Storage Conditions | Link |
Media | ||||||
LB | Luria Broth (Miller's LB Broth), Non-Sterile, 6.8 - 7.2, Molecular Biology Grade, suitable for regular E.coli culture, Powder | Sigma-Aldrich | L3522-1KG | $221 | 4C to 30C | [link] |
IPTG | Isopropyl β-D-thiogalactoside (IPTG), Powder, ≥99% (TLC), ≤0.1% Dioxane | Sigma-Aldrich | I6758-1G | $89.90 | -25C to -15C | [link] |
Kanamycin | BioReagent Kanamycin sulfate,
≥750 ug/mg, From Streptomyces kanamyceticus, Suitable for cell culture,
Suitable for plant cell culture, Powder | Sigma-Aldrich | K1377-1G | $47.70 | 4C to 30C | [link] |
Culture Tubes | ||||||
Culture tubes | Culture Tube, PS, 14mL, 18x95mm, Sterile, TC Treated, w/ Snap (Vent) Cap | Greiner Bio-One | 191160 | $258.15 | 4C to 30C | [link] |
Flasks | ||||||
250 mL baffled flasks | PYREX® 250 mL Delong Shaker Erlenmeyer Flask with Baffles | Pyrex | 4444-250 | $188.26 | 4C to 30C | [link] |
flask closures | Chemglass Life Sciences Closure, 38mm, Stainless Steel | Chemglass Life Sciences | Chemglass Life Sciences Closure, 38mm, Stainless Steel | $100.75 | 4C to 30C | [link] |
Prerequisite Protocols
Prepare selective media and inducer: 
Make Protein Purification Buffers and Media.
Make Protein Purification Buffers and Media.
Protocol
Prep overnight cultures.
Add 5 mL LB + kanamycin (50 ug / mL) to 15 mL culture tubes. Label each tube.
Poke your expression strain working stock with a pipette tip.
Eject the whole tip into each culture tube.
Incubate cultures overnight at 37C / 250 rpm for between 10 hrs and 16 hrs.
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Perform bulk outgrowth.
Back dilute overnights 1:1000 into fresh media (e.g., add 100 uL of overnight and 100 mL LB with Kanamycin to 250 mL Erlenmeyer flasks).
Incubate back diluted cultures at 37C / 250 rpm / to mid-log phase (OD600 between 0.4 and 0.6 ~ 3.5 hrs.)
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Induce protein expression.
Once bacterial cultures reach midlog phase (OD600 between 0.4 and 0.6) add IPTG to 500 uM. To do this, we added IPTG from 0.5M aliquots (1000x) at 1:1000.
E.g., for 100 mL of culture, we add 100 uL of IPTG at 0.5M.
Incubate induced cultures at 37C / 250 rpm / 4 hr to allow cells to express proteins.
Centrifuge cells and freeze pellets.
While induced cultures are incubating, pre-chill centrifuge and rotor to 4C
Pour 50 mL of each culture into 50 mL centrifuge tubes and label each (2x50 mL tubes per 100 mL culture).
Centrifuge cultures at 3200 rcf / 4C / 30 min.
Decant supernatant and reserve pellets.
Store pellets at -80C and allow to freeze (at least overnight)
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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