PSF
•Ni-NTA purification (denaturing) in microtitre plates•
- Get 96-deep well plate with induced E. coli cells, prepared according to Protocol 1, from the -80°C freezer
- Thaw at room temperature
lyse cells
- Add 150 µl Buffer A (6 M guanidine-HCl, 0.1 M NaH2PO4, 0.01 M Tris, pH 8.0, adjusted with NaOH)
- Resuspend cells by vortexing
- Incubate at room temperature 30 min
remove cellular debris
- Centrifuge 30 min 6200 rpm (Sigma centrifuge, Qiagen rotor)
- Pipet supernatants into filter plate (e.g. Millipore Multiscreen MADVN6550)
- Filter lysates into new filter plate
bind protein to NiNTA agarose and wash
- Add 25 µl 50% NiNTA agarose equilibrated in Buffer A
- Close filter plate with adhesive tape or parafilm
- Shake vigorously for 1 h in microtitre plate shaker
- Remove liquid by filtration, immidiately proceed with next step
- Wash three times:
- add 100 µl Buffer C (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris, pH 6.3, check pH before use)
- close filter plate with adhesive tape or parafilm
- shake 5 min
- remove liquid by filtration
elute bound proteins
- Add 25 µl Buffer C containing 0.1 M EDTA
- Shake 10 min
- Collect eluates by centrifugation 2 min 2000 rpm into new microtitre plate
- Analyse by SDS-PAGE.
alternative elution for MALDI mass spectrometry
- Wash four times:
- add 200 µl 5 mM Tris-HCl pH 8.0 or 50 mM NH4HCO3
- remove liquid by filtration
- Add 100 µl 35% acetonitrile (ACN), 0.1% TFA
- Shake 10 min
- Collect eluates by centrifugation 2 min 2000 rpm into new microtitre plate.
© 2003 by V. Sievert, Konrad Büssow last changed 12 Sep 2006