I am trying to prepare my cells in order to perform a Western Blot on them. I was wondering if anyone has had any experience with creating protein lysates for these cells.
Malcolm Nobre
Place the cell culture dish on ice and wash the cells with cold PBS. Aspirate PBS, then add ice-cold RIPA lysis buffer (1 mL per 107 cells/100 mm dish/150 cm2 flask; 0.5 mL per 5x106 cells/60 mm dish/75 cm2 flask). Scrape A549 cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube. You can also trypsinize the cells and wash with PBS prior to resuspension in RIPA lysis buffer in a microcentrifuge tube. Maintain constant agitation for 30 min at 4°C. Centrifuge in a microcentrifuge at 4°C. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet. Determine the protein concentration by Bradford assay or a bicinchoninic acid (BCA) assay. Bovine serum albumin (BSA) is a frequently used protein standard. Then perform Western Blot.
Hello Haley Luu
Please use the protocol given below.
Preparation of Protein Lysate from cultured A549 cells.
Preparation of RIPA lysis buffer (1X)
150 mM sodium chloride
1% NP-40 or Triton X-100
0.5% sodium deoxycholate
0.1% SDS (sodium dodecyl sulfate)
1mM EDTA
50 mM Tris, pH 8.0
You need to add protease and phosphatase inhibitor cocktail fresh in the RIPA buffer at the time of cell lysis to prevent proteolysis and dephosphorylation. The cells have to be kept on ice at all times.
I hope this will help.
Good Luck.
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Surajit Hansda
- Discard the medium in culture dishes with A549 cells and wash the cells using ice-cold PBS.
- Discard the PBS, add ice-cold lysis buffer.
- Scrape the cells using cold plastic cell scraper or by simply trypsinization. Collect the cells in microfuge tubes.
- Agitate the contents in microfuge tubes for 30 min at 4 °C.
- Centrifuge the tubes at 16000G for 20 min at 4 °C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.
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