Team:Washington/Notebook/IMAC protocol

BioRad Micro Column Protein Prep Protocol

• 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