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Cell Lysis

Written by Super User. Posted in Protein

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Cell Lysis Protocol For Western Blotting

Cell Lysis

Lysing Cells For Western Blotting

MATERIALS NEEDED:

Solubilizing Buffer
PMSF 100X
PBS 1X
Cell Lysis Buffer

METHOD (assume 6-well plates):

      • Remove media from all wells;  discard media.
      • Wash each well with 2mL 1X PBS once, touching pipette to sides (keep in dish). Eject from Pipetteman slowly (switch to slow ejection).
      • Add 500 uL of Cell Lysis buffer per well (if you want more concentrated protein samples, decrease this)
      • TIP! for western-blotting phosphorylated phospho-proteins: lyse directly in 100 uL SDS-PAGE Buffer.
      • Add 10 uL of 1XPMSF per well (protease inhibitor)
      • Add Trazylol 10uL per well (protease inhibitor)
      • Alternatively make a stock (master mix) solution of Cell lysis buffer, PMSF, and trasylol already added and add 520 uL to each well.
      • Label eppendorf tubes 2 mL or 1.5 mL
      • Get small needle syringe / scraper
      • Scrape wells first (this is not always neccessary - you can try just passaging with needle)
      • Then with syringe wash wells (5-)10X each - (5X is easier on the hands)
      • Remove all liquid carefully and put into eppendorf tubes
      • Centrifuge each tube for 2 min @ high speed

Also see our Western Blotting Membrane Stripping Protocol

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