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