Purification of GST-tagged FYVE proteins

• Transform BL21 E. Coli. and grow on Agar (+amp). There is a protocol for this step.

However check if there is a glycerol prep. of transformed bacteria already made- you can then use this and go to *.

• Next evening, pick a colony and grow overnight in 50ml LB/Amp, 37°C (shake at 250rpm).

• Put overnight culture into 2x500ml LB, add Amp.

• * Let grow until OD600=0.8 (2-3 hours)

• Add ZnCl2 to 1µM and IPTG (0.4mM) to induce protein production

• After 2.5 hours, pellet bacteria (Jouan centrifuge, 4,000rpm, 15 minutes, 4°C)

• Resuspend in <100ml of

PBS supplemented with 1µM ZnCl2, 150mM NaCl, protease inhibitor cocktail (lysis buffer)

Protease inhibitor cocktail ready made in freezer (G.07)- make sure you use the bacterial inhibitors.

• Transfer resuspended pellet into 2 x 50ml Falcon tubes and top up with lysis buffer

• Pellet as above, remove supernatant and snap freeze pellet with liquid nitrogen for protein prep the next day, or continue right through.

• Resuspend both pellets in 25ml total lysis buffer.

•Add lysozyme (1mg/ml), (make fresh, solid in freezer, general molecular biology box, G.O8) incubate for 15 minutes on ice.

• Freeze (1 min)-thaw with liquid nitrogen x2

• Add 10µg/ml DNAse, (stock in freezer, general molecular biology, G.O8) incubate on wheel/roller for 30 minutes, 4°C

• Pass suspension through 23G needle + syringe. Collect 50ml sample. (optional step)

• Spin in Kontron ultracentrifuge (TFT 55.38 rotor) at 33,000r.p.m. for 30 minutes

needs prior booking. Collect 50ml samples of supernatant and resuspended (to oiginal volume) pellet.

• harvest supernatant

• Wash 2.6 ml of a 75% slurry of glutathione-agarose beads X 3 with PBS (use 50ml falcon tubes, Jouan centrifuge) Use protocol sent with beads as a guide for next steps.

• Combine supernatant and beads in a 50ml Falcon tube, incubate at 4°C on wheel/roller for 1 hour. Collect 50ml sample of supernatant when done.

• Wash (resuspend and spin 5min 1000rpm, 4°C) the beads X3 with > 20ml PBS, 1µM ZnCl2, protease inhibitor cocktail.

• elute protein x 4 with 2ml 50mM Tris-HCl, pH 8.0, 10mM reduced glutathione, 1µM ZnCl2. Collect elutes and keep separately.

* analyse elutes (~20ml) and collected fractions (~4ml) by SDS-PAGE, 10% gel

• combine "pure" fractions and dialyse against 2x 1000ml of a suitable buffer- depending on envisaged use of the protein.

For lipid binding assays use

20mM HEPES-KOH (pH 7.6), 100mM KCl, 0.2mM DTT, 1µM ZnCl2