Purification of His₆-tagged proteins

• Innoculate 25ml LB + Amp starter culture with a single colony from a selective plate, 37°C o/n

• Innoculate 1 litre LB+Amp with 10ml starter culture, shake at 37°C until OD₆₀₀=0.6

• Induce expression with 0.1mM IPTG at 23-37°C for about 4 hours- use lower temperatures if degradation is a problem.

• Spin down bacs (at this point you can snap freeze with liquid nitrogen for later use)

Resuspend pellet in 30ml IMAC 5 + 0.5 mg/ml lysozyme incubate 10 minutes and then sonicate 10x5 second pulses on ice.

Alternatively

Add IMAC 5 + 0.5 mg/ml lysozyme + 0.1% TritonX100, leave on roller for 30 minutes at RT

for both methods also include bacterial protease inhibitors unless you are dealing with an enzyme (e.g myotubularin/UBPY/AMSH)!

• Spin lysate at 45,000 rpm in Optima centrifuge for 30 minutes at 4°C.

Add 1ml pre-washed (IMAC5) Ni-NTA beads to supernatant, leave on roller for 2 hours (or overnight)

•Spin down beads, take off supernatant. Wash beads x4 with IMAC 20

• elute with 7.5ml of IMAC 200

• Dialyse against 5 litres of 25mM HEPES/NaOH, pH 7.2, 150mM NaCl, 0.5mM DTT. First 2 hrs against 2.5 litres then overnight against 2.5 litres (4°C)

• Spin down any precipitate take supernatant and add 10% glycerol

• Determine protein concentration, check purity etc. by gels. Freeze aliquots by snap freezing on liquid nitrogen.

IMAC 5: 20MM Tris-Hcl pH 8.0, 300mM NaCl, 5mM imidazole

IMAC 20: 20MM Tris-Hcl pH 8.0, 300mM NaCl, 20mM imidazole

IMAC 200: 20MM Tris-Hcl pH 8.0, 300mM NaCl, 200mM imidazole