Cell Lysate

From OpenWetWare
Jump to: navigation, search

Protocols

Timepoint

  • At ??, cells were treated with NS (non-stimulated, 1st column of plate), 1,000 U(1uL) of IFNa (2nd column of plate), and 1,000U (1uL) of IFNg (3rd column of plate). Cells were then incubated for 0.5hr.

Sample Collection

  1. At ??? cells were placed on ice and collected for Western anlaysis. Protein lysates are stored in Box ?? of -20C storage.
    1. Cells and media are transferred to 1.5mL epindorfs and spun at 13,000 rpm, 4C, 15min.
        • Supernatant can be collected and aliquoted for testing here.
        • Otherwise, Supernatant is decanted and disposed of from epindorfs
    2. During spin, wells are washed and incubated with 0.5mL cold PBS.
    3. Well wash is transferred to epindorfs and washed once more with 0.5mL cold PBS
    4. Wells are washed with 0.5mL cold PBS.
    5. Wells are scraped with Cell Scrapper
        • 0.4mL transferred to epindorf, remaining liquid is left in chamber during scrape and then collected
    6. Epindorf is spun at 13,000 rpm, 4C, 15min.
    7. Supernatant is disposed of. As much as possible is removed.
    8. 60uL per epindorf of Cell Lysis buffer is added
      1. 10x Protease Inhibitor
      2. 10x Lysis Solution
      3. 100x Phosphates inhibitor
      4. remainder cold PBS
    9. Epindorfs are incubated on ICE for 20-30 minutes.
    10. Epindorfts are sonicated for 5secs at bottom and 5secs in liquid. Tip is washed between samples
    11. Epindorfts are spun at 13,000rpm, 4C, 15min.
    12. Protein lysates is collected into fresh epindorf tube that is labeled for long term storage at -20c.
Cell Line Sample Concentration 25ug Loading Buffer