Whole cell lysates (WCLs) of eukaryotic cells

Materials
	PBS 1X
	RIPA buffer
	Sepharose Beads

Procedure

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All following steps are performed in the cold room:

• Aspirate cell culture medium with a pasteur pipette
• Rince attaching cells carefully with cold PBS
• Add 1 mL RIPA buffer per 10 cm dish or 0.5 mL per 6 cm dish and incubate for approx. 2 min at 4°C
• Carefully detach the cells with a cell scraper and transfer the lysate in a fresh tube
• Mechanical shear the DNA by passing it multiple times through a fine needle
• Add 50 μL sepharose beads to the lysate
• Mix the suspension by overhead rotation for 5 min at 4°C
• Centrifuge lysate for 30min at 13.000 rpm, 4°C
• Transfer 100 μL of the supernatant into fresh tubes and mix 1:2 with SDS buffer, boil the sample briefly
• Store the remaining lysate at -80°C or mix 1:3 with RIPA buffer for the direct usage in a GST-Pulldown experiment