©CLONTECH	BIOL 340 Biochemical Methods Experimental Protocols
Instructor:Marie Pizzorno Department of Biology Bucknell University Office: BB205 Phone: (570) 577-3084 pizzorno@bucknell.edu

Growing GST containing bacteria and inducing expression with IPTG

1. Dilute overnight culture 1:8 into LB media containing 25 ug/ml of ampicillin (stock Amp solution is stored at -20oC and is 25 mg/ml).
2. Grow with vigorous shaking at 37oC for 3-4 hours. The OD (600nm) should be between 0.6 and 0.8.
3. Remove 1 ml of culture to microfuge tube, spin for 30 secs, remove supe and store pellet at -20oC This is the pre-induction sample.
4. Add IPTG to a final concentration of 0.25 mM (stock is 0.25M and stored at -20oC).
5. Continue shaking at 37oC for 3-4 hours (longer is OK, up to 5 hours). Save another 1ml sample as in step 3 (post-induction sample).

Harvesting GST proteins from bacterial cells

1. Harvest bacterial cells by centrifuging at 3-5 K rpm for 10-15 minutes. Pour off supe and let pellet drain.
2. Resuspend bacteria in 5 mls of harvest buffer (40mM Tris pH 8.0, 100mM NaCl, 5mM EDTA, 1% Triton X-100) and include protease inhibitors just before use.
3. Sonicate twice for 30 seconds each time, keeping bacteria cold during sonication. Color of solution should change from cream color to tan/brown color as bacteria lyse. Try to avoid foaming of the solution.
4. Spin lysate at 10,000 rpm for 20 minutes at 4oC.
5. Remove supe to clean tube avoiding any of the pellet. Add glycerol to final concentration of 10%. Aliquot into prechilled microtubes and freeze at -70oC.

Washing Glutathione-Sepharose Beads

1. Thoroughly but gently mix container of beads until it is a homogenous mixture.
2. Pipette 200 ul into microtube. Spin 30 secs.
3. Carefully remove most of supe with pipettor.
4. Wash beads 2-3 times with 1 ml of TNTG (40mM Tris pH 7.5, 100mM NaCl, 10% glycerol, 1% Triton X-100).
5. Remove final supe and resuspend beads in 200 ul of TNTG. Store at 4C.

Binding of GST proteins onto glutathione beads/Pull Down Assay

1. Place 200-400 ul of bacterial supe into chilled clean microtube. Add 25ul of mixed washed glutathione-sepharose beads.
2. Rock tubes at 4C for 30-60 minutes.
3. Spin. Carefully remove supernatant
4. Wash beads 2-3 times with 1 ml of TNTG.
5. Carefully remove as much as possible of final supernatant using micropipette or pulled Pasteur pipette if necessary.
6. If GST proteins are to be checked on SDS-PAGE then add 20-30 ul of protein sample buffer. Immediately incubate tubes at 95C for 3-5 minutes.
7. Place samples on ice or store at -20C until samples are loaded on SDS-PAG.
8. If GST proteins are to be used for binding assay, then add diluted brain extract and rock tubes at 4C for 1-2 hours.
9. Wash pellet 2-3 times in TNTG and then boil in 20-30 ul of protein sample buffer.
10. Store samples at -20C for electrophoresis on SDS-PAGE.