Home Support Protocols
- Solutions and Reagents
- Typically use RIPA buffer (25 mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Proteinase inhibitor cocktail should be added fresh before each use.
- The usage of SDS depends on the nature of the cells. For some cell lines, 0.1% SDS will release DNA and thus make it hard to extract proteins out. In those cases, omit SDS
- Preparation of cell lysates
- Add ice cold lysis buffer (1ml per 100mm-dish or 107 cells, or adjust based on your specific requirements). Scrape off cells (for adherent cells still on plate) and resuspend cells. Collect cells in a centrifuge tube and agitate for 30 min at 4°C.
- Spin cells at 4°C for 20 min at 12000 rpm.
- Save the supernatant which is the cell lysates.
- Add normal serum or irrelevant antibody from the same species and isotypes as the IP antibody you will use. The amount should be at least 5-fold more than the amount you will use for IP. Incubate for 1 hr at 4°C.
- For 1 ml lysate, add 100 ul of proteins A or protein G beads slurry (50 ul solid bed volume), and incubate at 4°C for 30 min on a rotator.
- Spin down beads at 14000g for 5 min at 4°C.
- Save the supernatant which is the pre-cleared lysates.
- D. Immunoprecipitation (IP)
- Add IP antibody to the pre-cleared lysates. You will need to determine the best amount of antibody to use. As a starting point, you may use 1 ug antibody for every ml of lysates.
- Incubate for a certain amount of time (from 1 hr to overnight, depending on your specific conditions) at 4°C.
- Add 100 ul of protein A or protein G slurry (50 ul solid bed volume) to 1 ml lysate and incubate for 3 hr at 4°C on a rotator.
- Spin down beads, and remove supernatant.
- Wash beads 3 times with lysis buffer.
- Add SDS-PAGE sample buffer to beads. Boil and run gel.