Trichloroacetic acid

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- All cells supernatants are centrifuged to remove cellular debris and then subjected to trichloroacetic acid (TCA) precipitation to concentrate the proteins.	- Pellets are washed twice with 100% cold acetone.

Upstream tips
- All cells supernatants are centrifuged to remove cellular debris and then subjected to trichloroacetic acid (TCA) precipitation to concentrate the proteins.
Protocol tips
- Pellets are washed twice with 100% cold acetone.

Publication protocol

A total of 750,000 cells were seeded into each well of 6-well plates (Corning). C. pneumoniae was added to the cells at a MOI of 5. Supernatants were collected from uninfected cells (day 0 post-infection) and from infected cells at 1, 2, 3, and 4 days post-infection. All supernatants were centrifuged to remove cellular debris and then subjected to trichloroacetic acid (TCA) precipitation to concentrate the proteins. Briefly, one volume of 100% TCA was added to four volumes of clarified supernatants and incubated for 10 minutes at 4°C. After incubation, samples were centrifuged at 800 x g for 5 minutes and pellets washed twice with 100% cold acetone. Washed pellets were dried by heating at 95°C for 5-10 minutes. Dried pellets were resuspended in Pierce Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific Life Sciences, Rockford, IL, USA), boiled at 95°C for 5 minutes, and loaded onto 12% SDS-polyacrylamide gels.

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