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 (normally do the following)
+
*Express and induce protein in culture under appropriate conditions:
#grow an overnight culture in ~25 mL LB/Amp (+Chloramphenicol if needed) from a colony <2 weeks post transformation
+
#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 10 uM IPTG (optimize for each protein)
+
#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).
+
#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 ~0.5 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Pellet beads 5 min at 1000 RPM and remove buffer
+
#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 5 mL of PBS
+
#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 A280; concentrate if necessary and store at -20)
+
#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:
  1. grow an overnight culture in ~25 mL LB/Amp (with Chloramphenicol if using Rosetta cells) from a colony <2 weeks post transformation.
  2. add 5 mL overnight culture to 1L TB/Amp and grow at 37C
  3. 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)
  4. let grow O/N at <25C (optimize induction time/temp for each protein, see Induction Conditions).
  5. centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point.

Lysis and Purification

  1. 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
  2. French Press cells 2 x 15 000 psi (see French Press Protocol)
  3. Centrifuge lysate at >15 000 RPM (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify
  4. 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
  5. Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads
  6. Incubate with rotation for 1h at 4C
  7. Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads
  8. Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 10 mL of PBS
  9. Prepare elution buffer containing 50mM Glutathionie in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8.
  10. Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay
  11. Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer
  12. Measure protein concentration (Bradford Assay or Quantification by Absorbance at 280nm; concentrate if necessary and store at -20)
  13. 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