Team:Washington/Notebook/IMAC protocol

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BioRad Micro Column Protein Prep Protocol

  • 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: STORE
    • Spin down cells at 4000rpm for 20 min in 50mL falcon tubes, discard supernatant
    • Store cells in -20C until ready for purification
  • Day 3/4: Purification
    • Lysis (~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
      • 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)
    • Purification (~1hr)
      • 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)
      • 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 YOU PURIFIED PROTEIN!
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