Cell Lysate

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.
 * 2) Cells and media are transferred to 1.5mL epindorfs and spun at 13,000 rpm, 4C, 15min.
 * 3) * Supernatant can be collected and aliquoted for testing here.
 * 4) *Otherwise, Supernatant is decanted and disposed of from epindorfs
 * 5) During spin, wells are washed and incubated with 0.5mL cold PBS.
 * 6) Well wash is transferred to epindorfs and washed once more with 0.5mL cold PBS
 * 7) Wells are washed with 0.5mL cold PBS.
 * 8) Wells are scraped with Cell Scrapper
 * 9) *0.4mL transferred to epindorf, remaining liquid is left in chamber during scrape and then collected
 * 10) Epindorf is spun at 13,000 rpm, 4C, 15min.
 * 11) Supernatant is disposed of. As much as possible is removed.
 * 12) 60uL per epindorf of Cell Lysis buffer is added
 * 13) 10x Protease Inhibitor
 * 14) 10x Lysis Solution
 * 15) 100x Phosphates inhibitor
 * 16) remainder cold PBS
 * 17) Epindorfs are incubated on ICE for 20-30 minutes.
 * 18) Epindorfts are sonicated for 5secs at bottom and 5secs in liquid. Tip is washed between samples
 * 19) Epindorfts are spun at 13,000rpm, 4C, 15min.
 * 20) Protein lysates is collected into fresh epindorf tube that is labeled for long term storage at -20c.