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Immunoprecipitation and western blotting detection of proteins Immunoprecipitation application generally includes 1) amplify the detection sensitivity of protein using immunoprecipitation. 2) determine the molecular weight of the unknown protein. 3) analyze the post-translational modification of the protein. 4) study the protein-protein interaction through co-immunoprecipitation technique. 5) useful for enzymatic activity detection when combined with native gel electrophoresis.

Depending on the application of immunoprecipitation, cells lysis should be optimized. Different cell lysis buffer contains different combinations of detergents and salts that exert harsh to mild membrane solubility effects.

Taking immunoprecipitation of phosphotyrosine containing proteins as example, two different strategies can be applied to get similar results: 1. antiphosphotyrosine antibodies can be used to immunoprecipitate protein complex and followed by western blot detection using antibody against the specific protein. 2. a specific antibody can be used to immunoprecipitate the protein complex followed by western blot detection using antiphosphotyrosine antobody.

General protocol for immunoprecipitation and western blot:

1. Cell lysis buffer
PBS: NaCl 116mM, Na2HP04 12mM, KH2PO4 1.5mM
Cell lysis buffer:
a. NP40 lysis buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40 (NP40), 0.25% Na+deoxycholate, and 10 μg/mL BSA.
b. Triton x-100 lysis buffer: 10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 50 mM NaF, 30 mM Na4P2O7, 150 mM Na3 VO4, 5 mM ethylenediaminetetracetic acid (EDTA), and 1% Triton X-100.
c. 30 mM Tris-HCl, pH 6.8, 150 mM NaCl, 1% NP40, 0.5% Na+ deoxycholate, and 0.1% SDS.

Note: protease inhibitors should be made in aliquote and stored at -20c until use.
300 μg/mL phenylmethysulfonyl fluoride.
20 μg/mL aprotinin.
10 μg/mL leupeptin.
100 μM Na3 VO4

2. Cell lysis procedure
- wash cells 3x @ RT with PBS
- lyse cells on ice by adding ~300-500ul lysis buffer per p100 dish. Make sure lysis buffer covers all surface of culture dish. Incubate lysate on ice for 5 min to 30 min with gentle rocking. Scrape cells off using a rubber policeman and transfer to a 1.5ml Eppendorf tube. Centrifuge 12000rpm x 10min @ 4c.
- Determine protein concentration and amount needed for following immunoprecipitation.

3. Immunoprecipitation procedure
- Wash protein A/G sepharose beads x 3 times with cold lysis buffer. Resuspend in 1 x starting volume of lysis buffer.
- Preclear lysate with 50 ul protein A/G sepharose beads @ 4c for 20 min with gentle rotating. Briefly spin down beads and transfer precleared lysate to fresh EP tube.
- Add 1 ug of specific antibody that recognize protein of interests in lysate and mixing gently for 1 hour @ 4c.
- Add 50 ul of washed protein A/G sepharose beads and rotate gently for 40 min @ 4c.
- Spin down briefly. Wash 2x with PBS + 0.5% Triton X-100, 2x with PBS + 0.1% Triton X-100, 2x with PBS.
- Protein can now be eluted in 1x sample buffer followed by boiling denaturing for 3min.
- Store @ -20c until use.

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