John Mundy,Institute of Molecular Biology,Copenhagen,Denmark

1 liter cell prep

1)grow 20ml cells O/N37℃ dilute 50X into prewarmed LB,grow to 0.6 OD or about 1hr.Induce w/ 2mM IPTG (238mg/0.5l),grow 3hr,spin 6k GS3 10',freeze at 70℃

2)resuspend cells (from 500ml)in fliptop in 25 mls.UPB8.0,freeze lN2.

3)thaw at37℃ sonicate 30" high,freeze lN2,thaw.

4)spin RT 10' 15k ss34,respin in fresh tube 10' 15k ss34.

5)decant again to new tube,add 5ml 50% Ni-NTA resin,mix gently RT 30'.

6)spin,decant and wash resin 3x in 50ml UPB6.3 w/5' gentle shaking each time.

7)resuspend in 25ml UPB6.3,pour into column and elute to top with another 25ml.s

8)elute bound proteins 20ml w/ UPB6.3+250mM imidazole.

9)lN2 freeze and store at 70℃

UPB 1 liter

[final] stock ml/l

8M UREA solid 480g

10mM TRISHCl 8.0 or 6.3 1M 10

0.1M Na-PO4 0.2M pHed 500

1mM bme 14.27 70ml

250mM imidazol solid 0.34g/20ml.