Italics indicate optional steps especially useful for the analysis of untested proteins.
GROWTH AND HARVESTING OF BACTERIA
- Add 100 ul ampicillin to 100 ml LB. Inoculate and grow overnight.
- In the morning, add overnight broth to 900 ml prewarmed LB. Grow 1 hour or to A600 greater than 0.5. Ampicillin can be added to help maintain the plasmid. Make glycerol stock from overnight culture if needed!
- Add 1 ml 0.1M IPTG (final 0.1mM) to the 1 L culture. Grow 3 to 5 hours or to A600 greater than 1.0. Prior to induction, remove 100 ul cells and add 100 ul SDS-PAGE sample buffer. Boil briefly and load 10 ul for SDS-PAGE. Sharper bands can be obtained if you pass through syringe several times to fragment DNA. Collect hourly samples to determine optimal induction time.
- Pour culture into two 500 ml centrifuge bottles and pellet at 5K 10' 4C.
- Resuspend in 10 ml resuspension buffer. Keep bugs on ice as much as possible from here on out to minimize proteolysis.
- Lyse bacteria by passing through the French Press 2x @12,000psi. Add PMSF if desired just before pressing. Save 100 ul of pressed lysate. Pellet 10K 10'. Load 2.5 ul supernatant for SDS-PAGE. Also resuspend pellet and load 2.5 ul to check whether the protein is insoluble.
- Transfer pressed lysate to a 15 ml centrifuge tube. Pellet cellular debris at 10K 10' 4C. Aliquot supernatant and store at -70C.
BINDING TO BEADS AND THROMBIN CLEAVAGE
- Incubate soluble lysate with 1/2 volume of 50% GST beads at 4C rocking on the Nutator for 30-60 minutes. 50 ml conical Falcon tubes work well.
- Pour into a BioRad Column and drain lysate. Save 100 ul of depleted lysate. Load 2.5 ul for SDS-PAGE.
- Wash beads with 3 column volumes resuspension buffer and 1 volume thrombin cleavage buffer.
- Seal the bottom of the column. Add 1 column volume thrombin cleavage buffer plus thrombin. 10 ug per mg fusion protein is a good starting point. Incubate 30-60 minutes at room temp rocking on the Nutator. If the protein is subject to degradation, try a 60 minute cleavage at 4C.
- Drain BioRad columns. Soluble cleaved fusion protein will be in the filtrate. Add a column volume of buffer to rinse out all cleaved protein. Rinse and save labelled tubes for re-use. Save about 20 ul of beads. Load 1-10 ul for SDS-PAGE.
- Optional: a second thrombin cleavage in 1/2 the previous volume.
- Inactivate thrombin with AEBSF or PMSF. Concentrate on the Centricon or Cell Stirrer if needed, or freeze as is. Save 100 ul of unconcentrated, purified protein. Load 1-10 ul for SDS-PAGE. Quantitate by comparison to 2.5, 1.0, and 0.25mg BSA standards on the same gel.
BINDING TO BEADS AND THROMBIN CLEAVAGE
Alternate Mini Prep
- Thaw out 1 ml aliquot of fusion protein lysate. Centrifuge to remove precipitate.
- Incubate 1 ml lysate with 0.4 ml 50% GST beads. Incubate at 4C with mixing for 1 hour.
- Centrifuge 5K 2' in 1.5 ml eppendorf tubes. Wash the beads 4X with PBST (0.01 % bME optional) and 2X with thrombin cleavage buffer. Stop here if protein bound to beads is needed for binding study.
- Resuspend the beads in 150 ul thrombin cleavage buffer and add 1.4 ug thrombin . Incubate at room temperature with mixing for 1 hour.
- Recover the supernatant containing the fusion protein by centrifugation. Stop the reaction with 0.7 ul PMSF. Incubate at room temperature 15'. Add another 0.7 ul PMSF and incubate another 15'.
- PBST: PBS (Phosphate-Buffered Saline) + 0.05% Tween20
- Resuspension Buffer: PBST + 2mM EDTA + 0.1% bME
- Thrombin Cleavage Buffer: Any buffer between pH 7-8.5 with Ca++ is adequate.
Try 50mM TRIS pH 8.0 + 150mM NaCl + 2.5mM CaCl2 + 0.1% bME
- 50% Glutathione Agarose Beads ("GST Beads")
1.85g beads per 50ml: Rehydrate beads in large volume of water for at least an hour. Wash with several volumes PBST through Whatman filter on a Buchner funnel to remove maltose stabilizer. Collect gel and add equal volume of PBST. Store at 4C. Do not freeze - beads are fragile and ice crystals will destroy.
- Ampicillin: 1.0g/10 ml sterile H2O, filter 0.22mM, store at -20C in 1ml aliquots. Use 1:1000.
- IPTG: 0.1M stock solution: 0.238g/10ml sterile H2O, store at -20C in 1ml aliquots
Alternate 5x solution: 5g/42ml
- PMSF: 0.3M stock solution in DMSO; use 1:1000
- Kanamycin: 0.25g/10ml, use 1:1000 only for cells with [pREP-4] plasmid!
- Sigma: Glutathione beads, cat # G-4510
- Sigma: Thrombin, 3560 NIH units/mg protein, cat # T-6759
- ICN: AEBSF: #193503
- bME should be freshly added when called for, as it is rapidly oxidized.
- PMSF should be added just prior to lysis of bacteria, as it is unstable in aqueous solutions.
- Instead of PMSF try less-toxic AEBSF. Stock solution: 0.2M in H2O, use 1:100
- nSec1, a sticky protein, gives a higher yield if 0.5M NaCl is added to the resuspension buffer and 0.05% Tween20 to the Thrombin Cleavage Buffer. Since the resuspension buffer is already 150mM NaCl just add 0.35M NaCl. If you intend to freeze the pressed lysate, leave in the cell debris. Centrifuge it out prior to purification.
- Stirred Cell Concentrator: Soak new filter in ddH20 1 hour. Store at 4C in 20% ethanol.