Purification of GST-fusion proteins
Procedure
The frozen bacteria pellets are thawed in 5 ml T-buffer on ice and resuspended. Afterwards the samples are incubated with 1 ml lysozyme for 30 min. The cells are broken through the addition of 20 ml lysis buffer and the treatment of ultrasonic sound (impulse 50%, 20sec, 3-5x) on ice water.
300 µl sepharose-solution is added to the suspension and incubated for 10 min with head-over-head rotation. Afterwards the lysate is centrifuged for 20 min at 16000 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.