Coimmunoprecipitation (whole cell lysates)

1. Lyse cells on ice for 5 min. then spin at 20 000g for 5 min. at 4C.

2. Mix 40ul Protein A agarose beeds, 10ul antibody and 0.5ul lysis (EB or NET) buffer.

3. Rotate 1h at 4C, then spin at 20 000g for 5 min. at 4C.

4. Wash in 1ml lysis buffer, then spin at 20 000g for 5 min. at 4C.

5. Add 1ml cell lysate, rotate for 4-6h, then spin at 20 000g for 5 min. at 4C.

6. Wash in 1ml lysisbuffer, then spin at 20 000g for 5 min. at 4C(repeat this step 2-3 times)

7. Resuspend in 10ul 6x protein sample buffer, heat for 5 min. at 95C, spin at 20 000g for 5 min. Move supernatant to new tube and use it for SDS PAGE.

EB: 20mM Tris pH7.0, 500mM NaCl, 10mM EDTA, 20mM glycerophosphate (optional), 20mM NaF,0.2% Triton X100, 2mM sodium pervanadate, 1mM PMSF, 30 ug/ml aprotinin;

NET: 20mM Tris-Cl, pH8.0, 200mM NaCl, 1mM EDTA, 0.1% Nonidet P-40, 10% glycerol, 2mM sodium pervanadate, 1mM PMSF, 30 ug/ml aprotinin;




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