Immunoprecipitation from transfected cells

Drain medium and gently wash cells once with ice-cold PBS (0.5ml for mw6; 1 ml for 6-cm dish; 3 ml for 10-cm dish).

Use a gentle stream to dislodge cells with PBS.

Centrifuge the tube at 3000 x g for 1 min and discard the supernatant.

Resuspend pellet of cells with 500 μl of ice-cold IP buffer

Lyse the cells by sonication: 3 times 10’’/30% on ice, waiting 1 minute in between for the lysate to cool down

Incubate the whole cell lysate on an end-to-end rocker at 4°C/30’ to solubilize the membrane proteins.

Spin down at 14,000 x g for 10 minutes at 4°C to pellet debris and genomic DNA, save the supernatant.

Save 75 µl, Cell Lysate (CL) and add 25 µl of SB4X

Mix total proteins (equal amounts for each sample) with 10-20 µl of Protein G beads and 2 µg of specific antibody. Note: wash Protein G Beads 3 times with PBS to remove ethanol present in the storage buffer. Let equilibrate in IP buffer for minute before use.

Incubate on an end-to-end rocker at 4°C/overnight

Separate the beads and save 75 µl of Flow Through (FT) and add 2 µl of SB4X

10. Wash beads 3 times with 1 ml of IP buffer

11. Elute with 50 µl of SB1X (IP)

12. Warm samples at 42°C/10’ (membrane proteins would start to aggregate at higher temperature).

13. Load 10 µl of each sample per well for SDS-PAGE