| John Mundy, Institute of Molecular Biology, Copenhagen, Denmark
http://www.arabidopsis.org/info/Protocols_Mundy2.jsp#his
1 liter cell prep
1) grow 20ml cells O/N 37 C, 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 C.
2) resuspend cells (from 500ml) in fliptop in 25 mls. UPB8.0, freeze lN2.
3) thaw at 37 C, 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 C.
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.
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