Team:Washington/Protocols/50mLPurificationCapD

BioRad Micro Column Protein Prep Protocol

Day 1: OVERNIGHTS

• Pick a single colony from plate and inoculate 2mL TB/LB +Kan in a 14mL culture tube
• Shake at 37deg 16-24hrs

Day 2: EXPRESSION

• Inoculate a STERILE 250mL flask that contains 50mL of TB+Kan media with 1mL (or 500µL of overnight if you used TB) of the overnight.
• Grow at 37 degrees until OD600 reaches 0.4-0.6 (generally 2-4 hrs)
• Add IPTG for final concentration of 0.5mM.
• Transfer to desired expression temperature and let express for the appropriate amount of time:
  • 22C = 16-24hrs
  • 30C = 8-12hrs
  • 37C = 4-6hrs

Day 3: STORE

• Spin down cells at 4000rpm for 20min in 50mL falcon tubes
• Pour off supernatant
• Store cells at -20C until ready for purification

Day 3/4: PURIFICATION

• Lysis (~1hr) KEEP ON ICE OR IN COLD ROOM AS MUCH AS POSSIBLE
  • Add 500uL of wash buffer and vortex to resuspend cells
  • Add 1mL of lysis buffer and gently pippette up and down (minimizing bubbles)
  • Transfer to a 2mL eppindorf tube.
  • Continue to incubate at room temp for 20min, mixing with plate mixer.
    • If worried about protein stability then incubate in the cold room for 1hr on rocker.
    • Save 50uL of lysis if want to run on gel.
  • Spin the lysis for 30-60min at 15000rpm
    • Spin longer if supernatant is not clear enough, but one hour should really be sufficient!
  • Transfer supernatant to a fresh tube (~1800uL) by decanting or careful pipetting

• Purification KEEP ON ICE OR IN COLD ROOM AS MUCH AS POSSIBLE
  • Bind Protein (~1/2hr)
    • Add 200uL of TALON/NiNTA Agarose 50% slurry to the column and place in 2mL centrifuge tube
      • Use a 1000uL tip otherwise the beads get stuck!
      • Final of 100uL of actual beads
    • Spin at 2000rpm for 2min
    • Discard flow-through and replace bottom cap
    • Add 500uL of supernatant, cap the top of the column, and mix (DO NOT VORTEX)
    • Let gently incubate on rocker for 5min
    • Remove caps and place in 2mL centrifuge tube
    • Spin 2000rpm for 2min
    • Discard flow-through and replace bottom cap
    • Repeat until all supernatant has passed through (~3-4x)
  • Washing Protein (~1/2hr)
    • Cap the bottom, add 500uL wash buffer, cap the top, mix, agitate 5min gently, remove caps and place in 2mL tube, spin 2000rpm 2min, discard flow-through
    • Cap the bottom, add 500uL wash buffer, cap the top, mix, agitate 5min gently, remove caps and place in 2mL tube, spin 2000rpm 2min, discard flow-through
    • Cap the bottom, add 500uL wash buffer, cap the top, mix, agitate 5min gently, remove caps and place in 2mL tube, spin 2000rpm 2min, discard flow-through
  • Eluting Protein (~1/4hr)
    • Cap the bottom, add 200uL of elution buffer, cap the top, mix, agitate 5min gently, remove caps and place in FRESH Eppendorf tube, spin 2000rpm 2min
    • THE FLOW-THROUGH IS YOUR PURIFIED PROTEIN!

Wash Buffer (GENERAL STOCK BUFFER):

• 50mM pH 7.4 HEPES
• 500mM NaCl
• 25mM Imidazole

BUGBUSTER Lysis Buffer:

• 50mM pH 7.4 HEPES
• 500mM NaCl
• 25mM Imidazole
• 2x Bug Buster
• 2mg/ml lysozyme (small scoop)
• 0.2mg/ml DNAse (small scoop)

Elution Buffer:

• 50mM pH 7.4 HEPES
• 500mM NaCl
• 500mM Imidazole