Immunoprecipitation (IP). Solutions and Reagents Lysis buffer:. 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.

Pre-clearing. 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.

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Incubate for 1 hr at 4°C. Download bigo live for mac. 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. 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.

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Boil and run gel.