Protocol for harvesting phusion protein.

Materials needed:

Glycerol stock of transformed E. coli cells expressing the His-tagged protein of interest.
The strain used is named EQ458. It is located in common species box; this is a Rosetta 2 (DE3) E. coli strain containing 6his-phusion-pET28 plasmid. The plasmid is KanR and the strain itself is CamR. The frozen stock is overnight growth of single colony. link to strain

LB medium. LB recipe
Chloramphenicol stock. Cam recipe
Kanamycin stock. Kan recipe
Refrigerated centrifuge.
FRENCH laboratory press.
IPTG, 100 mM stock. Dissolve 2.38 g IPTG in 100 mL deionized water. Filter sterilize and store at -20 C.
Ni-NTA resin spin column. ThermoFisher HiPur Ni-NTA spin columns, 1ml (cat # 88225).
30k cutoff Amicon ultra-filtration membrane. Amicon Ultra-15 centrifugel filter units from Millipore (cat # UFC903008).

Lysis Buffer:

  • 50 mM NaH2PO4
  • 300 mM NaCl
  • 10mM Imidazole
  • Adjust pH to 8.0 using NaOH

Wash Buffer:

  • 50 mM NaH2PO4
  • 300 mM NaCl
  • 20mM Imidazole
  • Adjust pH to 8.0 using NaOH

Elution Buffer:

  • 50 mM NaH2PO4
  • 300 mM NaCl
  • 250 mM Imidazole
  • Adjust pH to 8.0 using NaOH

Polymerase storage buffer:

  • 50% Glycerol
  • 100 mM Tris/HCl pH 8.0
  • 0.2 mM EDTA
  • 2 mM DTT
  • 0.2% NP-40; nonionic detergent
  • 0.2% Tween20

Procedure: (for 2 x 500 mL culture)

  • Streak LB plate supplemented with Kan and Cam using frozen stock. Growth overnight at 37 C.
  • Inoculate 1.5 mL of LB broth supplemented with Kan and Cam with single colony from streak plate.
  • Grow overnight at 37 C shaking at 200 rpm (????).
  • Use x uL of overnight culture to inoculate 500 mL of supplemented LB broth, grow as before for 3-4 hours until logarithmic phase is reached. Also, set up a control culture which will serve as an uninduced control.
  • Induce the cultures to express proteins by adding IPTG at a final concentration of 1.0 mM (? is this correct?) followed by overnight growth (what is total time) at 18 C.
  • Collect cells by centrifugation. Conditions as follows: 4 C, at 10,000 x g for 15 mins.
  • Resuspend with 3 mL of lysis buffer and place tubes in cold room bath shaker (at dial 6 ?????) for ~ 2 hours.
  • French press; use the full cell holding (10 mL - 35 mL), and use 1500 psi pressure.
  • Lyse cells, heat denature at 70 C for about 15 mins.
  • Spin down, 3700 - 4000 x g for 15 mins.
  • Collect and keep supernatant (SN) for IMAC purifications.

IMAC purification:

  • Prepare a 1 mL Ni-NTA resin column.
  • Saturate column with 5x lysis buffer.
  • Bind with 1:1 lysis buffer:SN sample.
  • Wash with 5x wash buffer.
  • Elute with elution buffer and collect samples after the first 1 mL flow through.
  • Use 30k cutoff Amicon ultra-filtration membrane (???how do you use this???).
  • Top-off with storage buffer.
  • Spin at 4000 x g for 2.5 hours or so until volume is close to ~ 1 mL.
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Contributors to this topic Edit topic JeffreyBarrick, CraigBarnhart, SimonDAlton
Topic revision: r1 - 2014-04-11 - 20:08:35 - Main.CraigBarnhart
 
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