Purification of GST-fusion proteins
LB-Medium 10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,
ad 1l A.bidest
Ampicillin 100μg/ml
IPTG 100mM Isopropylthiogalactosid
T-buffer 100mM Tris (pH 8.0), 5mM EDTA
Lysis buffer (200 ml): 10 ml Tris pH 8.0 [1M] → 50 mM
4 ml EDTA [0.5 M] → 10 mM
30 ml NaCl [1M] → 150 mM
40 ml Glycerol [50 %] → 10 %
0.5 ml Triton [10 %] → 0.025 %
add freshly à 20 ml:
50 μl DTT [1M] → 2.5 mM
20 μl PMSF [10 mg/ml]
20 μl Leupeptin [10 mg/ml]
20 μl Aprotinin [10 mg/ml]
20 μl Pefablock [10 mg/ml]
Lysozyme 1 mg/ml
Sepharose- solution,
Glutathione-sepharose-solution
PBS (1x) 24g NaCl, 0.6g KCl, 3.45g Na₂HPO₄ x 7 H₂O, 0.6 g
KH₂PO₄, pH 7.4, ad 1l mit A.bidest.
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.