Team:Washington/Protocols/50mLPurificationCapD

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== 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!
 +
 +
 +
-------------------------------''Buffer Examples; alter to fit your protein’s ideal buffer'' -------------------
 +
'''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
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Revision as of 18:57, 14 September 2010

A Graphical Overview Will Go Here

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!


Buffer Examples; alter to fit your protein’s ideal buffer -------------------

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

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