Difference between revisions of "Purification of GST Fusion Proteins"
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==Bacteria Production and Induction== | ==Bacteria Production and Induction== | ||
| − | + | *Express and induce protein in culture under appropriate conditions: | |
| − | #grow an overnight culture in ~25 mL LB/Amp ( | + | #grow an overnight culture in ~25 mL LB/Amp (with Chloramphenicol if using Rosetta cells) from a colony <2 weeks post transformation. |
| − | #add 5 mL culture to 1L TB/Amp and grow at 37C | + | #add 5 mL overnight culture to 1L TB/Amp and grow at 37C |
| − | #grow to OD600 of 0.6-1.0 and induce with | + | #grow to OD600 of 0.6-1.0 and induce with 100 uM IPTG (the concentration of IPTG and duration of induction should be optimized for each protein) |
#let grow O/N at <25C (optimize induction time/temp for each protein, see [[Induction Conditions]]). | #let grow O/N at <25C (optimize induction time/temp for each protein, see [[Induction Conditions]]). | ||
| − | #centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point | + | #centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point. |
| + | |||
==Lysis and Purification== | ==Lysis and Purification== | ||
#Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet). Freeze in liquid nitrogen if not continuing with purification | #Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet). Freeze in liquid nitrogen if not continuing with purification | ||
#French Press cells 2 x 15 000 psi (see French Press Protocol) | #French Press cells 2 x 15 000 psi (see French Press Protocol) | ||
| − | #Centrifuge lysate at >15 000 RPM for 30 min to clarify | + | #Centrifuge lysate at >15 000 RPM (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify |
| − | #Add | + | #Add 1.0 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Let sit for 30 min. to allow beads to settle or pellet beads for 5 min at 1000 RPM and remove buffer |
#Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads | #Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads | ||
#Incubate with rotation for 1h at 4C | #Incubate with rotation for 1h at 4C | ||
#Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads | #Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads | ||
| − | #Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another | + | #Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 10 mL of PBS |
| + | #Prepare elution buffer containing 50mM Glutathionie in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8. | ||
#Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay | #Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay | ||
| − | #Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) | + | #Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer |
| − | #Measure protein concentration (Bradford or | + | #Measure protein concentration ([[Bradford Assay]] or [[Quantification by Absorbance at 280nm]]; concentrate if necessary and store at -20) |
| − | #Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and protein (0.5-10 ug) by SDS-PAGE | + | #Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and purified protein (0.5-10 ug) by SDS-PAGE |
| + | [[Category:Protein Purification]] | ||
Latest revision as of 14:40, 12 August 2009
Bacteria Production and Induction
- Express and induce protein in culture under appropriate conditions:
- grow an overnight culture in ~25 mL LB/Amp (with Chloramphenicol if using Rosetta cells) from a colony <2 weeks post transformation.
- add 5 mL overnight culture to 1L TB/Amp and grow at 37C
- grow to OD600 of 0.6-1.0 and induce with 100 uM IPTG (the concentration of IPTG and duration of induction should be optimized for each protein)
- let grow O/N at <25C (optimize induction time/temp for each protein, see Induction Conditions).
- centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point.
Lysis and Purification
- Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet). Freeze in liquid nitrogen if not continuing with purification
- French Press cells 2 x 15 000 psi (see French Press Protocol)
- Centrifuge lysate at >15 000 RPM (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify
- Add 1.0 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Let sit for 30 min. to allow beads to settle or pellet beads for 5 min at 1000 RPM and remove buffer
- Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads
- Incubate with rotation for 1h at 4C
- Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads
- Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 10 mL of PBS
- Prepare elution buffer containing 50mM Glutathionie in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8.
- Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay
- Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer
- Measure protein concentration (Bradford Assay or Quantification by Absorbance at 280nm; concentrate if necessary and store at -20)
- Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and purified protein (0.5-10 ug) by SDS-PAGE
