WB Conditions: Blocking: 5% milk in TBS/0.1% Tween 20, 60 min, RT, rocking platform. GFAT Ab (3rd or 2nd bleed): 1 μg/ml in 5% milk in TBS/0.1% Tween 20. Incubate on a screw platform for Falkon tube for overnight, 4°C. Washing: 3x10 min with TBS/0.1% Tween, rocking platform. Secondary Ab: anti-sheep HRP-labeled IgG (Pierce), 1:3000 in 5% milk in TBS/ 0.1% Tween 20, 1h, RT, rocking platform. Hemiluminescence detection: ECL Western Blotting Detection Reagents (Amersham, GE Healthcare, #RPN2106).
IP Conditions: Cell lysate containing recombinant GFAT1: 1-1.5 mg (top volume with lysis buffer up to 500 μl). GFAT1 Ab (3rd bleed): 3 μg/lysate. 50% Proten G-sepharose: 10 μl/lysate. Incubate for 2h on a rotating wheel, 4°C. Spin at 6000 rpm, 1 min. Wash twice with Lysis buffer + 0.5 M NaCl and twice with Buffer A, recovering beads by spinning at 6000 rpm, 1 min between washings. Add equal volume of 2x sample buffer +DTT and boil off any bound to the beads proteins at 95°C for 5 min. ￼￼Spin at 13000 rpm, 5 min. Analyze supernatants by SDS-PAGE and Western blot analysis.