Preparation of deterg.-resist. membrane microdomains (Sucrose Gradient)

Materials
Homogenisation buffer	50 mM Tris pH 7.4	[0.5M]	▶ 10 ml
	2 mM MgCl2 8 % sucrose	[1M]	▶ 200 µl ▶8 g
	ddH2O		▶ 89.8 ml
TNE buffer	20 mM TrisHCL pH8 [1M]		▶ 2 ml
	130 mM NaCl [5M]		▶ 2.6 ml
	5 mM EDTA [0.5M]		▶ 1 ml
	10 µg/ml Aprotinin [5mg/ml]		▶ 200 µl
	10 µg/ml Leupeptin [10mg/ml]		▶ 100 µl
	1 µg/ml Pefabloc [1mg/ml]		▶ 100 µl
	1 mM PMSF [200mM]		▶ 500 µl
	0.5 % TritonX 100		▶ 500 µl
	ddH2O		▶ 92.9 ml
Sucrose	80% Sucrose: 8 g in 10 ml TNE
	35% Sucrose: 14 g in 40 ml TNE
	5% Sucrose: 1 g in 20 ml TNE

Procedure

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• Two days for experiment: Dissolving of sucrose in TNE at room temperature without protease inhibitors (80% needs 1 to 2 days), add inhibitors a short time before starting experiment
• Cells seeded on poly-L-lysin coated plates 1 day for experiment
• 1 h Crosslink with 1st antibody (1:1000) in 3 ml medium, 37 °C
• 3x wash with PBS
• 30 min 2nd antibody in 3 ml medium (1:500), 37°C
• 1x wash, 1 ml ice-cold homogenizing buffer on cells
• Scrape cells from plate, 15 min 7000 rpm, 4°C
• Supernatant + 4ml homogenizing buffer + 5mM EDTA (1:100) in ultracentrifuge tubes  balance out the tubes!
• 2h, 48 000 rpm, vacuum
• Discard supernatant
• Dissolving of pellet in 280 µl ice-cold TNE buffer for 10 min in cold room
• + 490 µl 80% sucrose (mixing)
• + 2500 µl 35% sucrose (careful: gradient!!!)
• + 800 µl 5% sucrose
• balance out the tubes!
• 18h 48 000 rpm, vacuum
• 8 fractions á 480 µl + 4x SDS buffer