Protein Structure Factory - E. coli protein expression

Prepare a soluble protein extract in a 96-well filter plate (Durapore MADV N 65, Millipore) according to Protocol 2. Each microtitre plate well contains about 150 µl extract.
Add 10 µl of 10% (settled bed volume) glutathion agarose equilibrated in 100 mM Tris-HCl, pH 7.4. For preparation of equilibrated glutathion agarose let 0.1 g dry agarose swell in 10 ml water for 2 hours, discard water, fill up with buffer up to 10 ml.
Shake for 30 min at room temperature (slow vortex).
Remove liquid by filtration on vacuum manifold
Wash twice with 200 µl of:
- 40 mM Tris-HCl, pH 7.4
- 0.2 M NaCl
Add 25 µl of 1xSDS-PAGE loading buffer and resuspend the beads in the filter plate by pipetting. Leave 5 min on the beads, then transfer to a 96-well microplate.
Denature 3 min at 95°C, and load 9 µl on the gel.

•Glutathion affinity purification in mircotitre plates•