ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! |- | |PBS 1X |- | |RIPA buffer |- | |Sepharose Beads |} === Procedure === ----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 s..."

2025-02-27T13:10:35Z

Created page with "{| class="wikitable" |+ !Materials ! |- | |PBS 1X |- | |RIPA buffer |- | |Sepharose Beads |} === Procedure === ----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 s..."

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{| class="wikitable"
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!Materials
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|PBS 1X
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|RIPA buffer
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|Sepharose Beads
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=== Procedure ===
----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

Whole cell lysates (WCLs) of eukaryotic cells - Revision history