Protein Structure Factory - E. coli protein expression - High throughput protein expression

Prepare a soluble protein extract in a 96-well filter plate (Durapore MADV N 65, Millipore) according to Protocol 2. Use only 0.1 mM EDTA for this extract. Each microtitre plate well contains about 150 µl extract.
Add 15 µl 0.1 M imidazole (end concentration 10 mM).
Add 25 µl of 20% NiNTA agarose (Qiagen) equilibrated in 50 mM Tris-HCl, pH 8.0.
Shake 30 min at room temperature.
Remove liquid with a vacuum manifold (Millipore).
Wash three times on the vacuum manifold with 200 µl Wash Buffer, shake in between. Wash buffer is removed with the vacuum manifold.
Wash buffer:
- 50 mM Tris-HCl, pH 8.0
- 0.3 M NaCl
- 20 mM imidazole
Remove all liquid by brief centrifugation.
Elute with 25 µl of the wash buffer containing 250 mM imidazole. Use brief centrifugation instead of the vacuum manifold to collect all eluate.
Analyse eluates by SDS-PAGE.

•NiNTA purification in mircotitre plates (non-denaturating, manually) •