Team:Washington/Notebook/IMAC protocol

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(Difference between revisions)
(BioRad Micro Column Protein Prep Protocol)
 
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*Buffer Examples (Alter to fit your protein's ideal buffer)
*Buffer Examples (Alter to fit your protein's ideal buffer)
 +
**Lysis Buffer
 +
***1.5x BugBuster (Solubilizes cell wall)
 +
***1mg/ml lysozyme (Helps break cell wall)
 +
***0.1mg/ml DNAseI (chews up DNA)
 +
***1mM PMSF (Protease Inhibitor)
 +
***1x PBS
 +
***30mM Imidizole
 +
**Wash Buffer
**Wash Buffer
***1x PBS
***1x PBS

Latest revision as of 02:52, 22 October 2009

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BioRad Micro Column Protein Prep Protocol


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  • Day 0: Clone gene of interest into vector of interest using standard cloning techniques.
  • Day 1: Overnights
    • Pick a single colony from plate and inoculate 1mL fresh media, with proper antibiotic, in 15mL culture tube
    • Place lid on tube as not to form an air tight seal
    • Shake at 37C for 16-24 hours at 200+ rpm
  • Day 2: Expression
    • Inoculate 50mL media with 500uL over night, in 250mL flask
    • Shake 250mL flask at 37C at 200+ rpm
    • Monitor OD600 of culture until OD600 ~0.4
      • Use 200uL of culture and 800uL water in 1mL cuvette
      • Read OD600 and multiply by 5 t get original OD600
      • Should take ~2-3 hours
    • INDUCE culture once culture gets to OD600 ~0.4 with IPTG so that the final concentration of IPTG is 0.5mM (ie 25uL 1M IPG for 50mL culture)
    • Place in shaker 24-36hr at 18C.
  • Day 3: Purify (~3.5 hours)
    • Pellet Cells (0.5hr)
      • Spin down cells at 4000rpm for 20 min in 50mL falcon tubes, discard supernatant
      • Cells can be stored in -20C until ready for purification
    • Lyse (0.5-1hr)
      • Add 1mL of lysis buffer, re-suspend, transfer to a 2mL eppindorf tube
      • Continue to incubate at room temperature for 20min, mixing with plate mixer
        • If worried about protein stability then incubate in cold room for 1hr on rocker
    • Pellet Insoluble Fraction (0.5-1hr)
      • Spin the lysis for 30-60min at 15000rpm (spin longer if supernatant is not clear, but 1hr should be plenty!)
      • Transfer supernatant to a fresh tube (~1800uL)
    • Filter (0.1hr)
      • Run supernatant through a 0.4micron filter to remove any remaning particules that would clog columns.
    • Bind Protein (0.5hr)
      • Add 100uL of NiNTA Agarose 50% slurry to each column (this is the Ni-beads)
      • Add 600uL of diH2O to each column
      • Spin 500rpm for 10s, discard flow through
      • Add 500uL of wash buffer, spin, discard flow through
      • Add 500uL of supernatant, spin, discard flow through
        • Repeat previous step until all supernatant has passed over column (~4x)
    • Wash Protein (0.5hr)
      • Add 500uL of wash buffer, spin, discard flow through
    • Elute Protein (0.2hr)
      • CHANGE TO FRESH COLLECTION TUBE
      • Add 200uL of elution buffer pipette up and down to mix the beads
      • Incubate for 2min the spin at 500rpm for 1min
      • THE FLOW THROUGH IS YOUR PURIFIED PROTEIN!
  • Recover the beads for re-use (~45min)
    • Extract beads using 200uL diH2O and transfer to 15mL falcon tube
      • Store columns in dry place for later use after thoroughly washed out with water. It is fine to just run tap water over the columns after beads have been removed
    • Spin down 800rpm for 5min, then decant supernatant
    • Fill falcon tube with 100mM EDTA, and re-suspend beads
    • Spin down 800rpm for 5min, then decant supernatant
    • Fill falcon tube with diH2O, and re-suspend beads
    • Spin down 800rpm for 5min, then decant supernatant
    • Fill falcon tube with 100mM NiSO4, and re-suspend beads
    • Spin down 800rpm for 5min, then decant supernatant
    • Fill falcon tube with diH2O, and re-suspend beads
    • Spin down 800rpm for 5min, then decant supernatant
    • Add 20% ethanol until a 50% slurry has been reached.
    • Store at 4C

  • Buffer Examples (Alter to fit your protein's ideal buffer)
    • Lysis Buffer
      • 1.5x BugBuster (Solubilizes cell wall)
      • 1mg/ml lysozyme (Helps break cell wall)
      • 0.1mg/ml DNAseI (chews up DNA)
      • 1mM PMSF (Protease Inhibitor)
      • 1x PBS
      • 30mM Imidizole
    • Wash Buffer
      • 1x PBS
      • 30mM Imidizole
    • BUGBUSTER Lysis Buffer
      • 1x PBS
      • 1mM PMSF
      • 2x Bug Buster
      • 2mg/mL lysozyme
      • 0.2mg/ml DNAse
    • Elution Buffer
      • 1x PBS
      • 250mM Imidizole