Learn about cell lysis
Cell Lysis Protocol For Western Blotting
Lysing Cells For Western Blotting
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
Our Protein Protocols