Protein Purification

RSB250-A Recipe:

• 20mM Tris
• 250mM NaCl
• 30mM Imidazole
• 10% Glycerol (v/v)

• 20mM Tris
• 250mM NaCl
• 500mM Imidazole
• 10% Glycerol (v/v)

Induction

1. Grow a small booster culture in LB Broth overnight (~2-10% of final culture volume) w/ appropriate antibiotics
2. Innoculate full scale culture & monitor OD600 every 15-30 minutes until OD600 reaches 0.8
3. At OD600=0.8, induce with appropriate concentration of inducer (e.g. 1mM final concentration of IPTG for lac promoter)
4. Allow to grow overnight @ 25°C

Pelleting & Sonication

1. Collect cultures by centrifugation for 30 minutes @4°C & 5000g
• If sample volume is too large, repeat centrifugation (discarding the supernatant after each centrifugation)
2. Resuspend pellet in RSB250-A (minimum 5% of culture volume)
3. Pellet must be completely dissolved (no cell clumps) for efficient lysis.
   • For especially difficult to resuspend pellets, sucking up and ejecting the cell suspension through a large gauge (narrow) needle several times can break up the pellet effectively
4. Sonicate 5 cycles
• 1 cycle consists of 15 of lysis (10W power) followed by 5 seconds of cool down
• Lysis must be done on ice or excessive protein degradation will occur
5. Spin lysate down @4°C & 20000g for 30 minutes
6. Transfer supernatant into 15 mL Falcon tubes
7. Add Ni-Agarose beads (1mL of beads per 100mL of culture volume)
• Wash beads 2-3 times with equal volumes of RSB250-A
• Spin down cells @ ~3000 rpm in a microcentrifuge
8. Rotate overnight in a 4°C cold room
9. Pour lysate and beads into a chromatography column, allow to empty through gravity flow
• Retain flow through (FT) on ice for later analysis
10. Wash with 10 mL RSB250-A two times
• RSB250-A must be cold
• Collect wash fractions for later gel on ice
11. Elute with 1% of culture volume of RSB250-B
• Collect elution fraction on ice
12. Eluted protein can be stored at 4°C or -80°C

Thawing Mammalian Cells

1. Prepare plates with 9mL of appropriate supplemented cell culture media
2. Take the cells out from the -80 freezer
3. Shake cells in a water bath at 37 degrees C
• Do this quickly, DMSO is extremely toxic to cells
4. Spray this with tons of ethanol, dry with kimwipe, and put this in TC hood
5. Add 1mL of media to the thawed cells, transfer immediately into plate
6. Take 1 mL of media from the plate and wash the tubes to get any remaining cells
7. Incubate overnight in cell incubator
8. Passage cells 1:2