CoIP protocol

Co-IP Procedure (from XGD)
(1) Preparing magnetic beads (for 6-well plate)

1. Aliquot 7 µl of Protein A beads (mouse antibody) to each 1.5 ml microfuge tube.
2. Wash beads once with 0.5 ml of Low salt wash Buffer; 4 °C, 4,000 g, 2 min, discard supernatant.
3. Resuspend beads with 0.5 ml Blocking Buffer, Block beads for at least 4 hours or overnight at 4 °C on a rotator.
4. Wash beads once with 0.5 ml of Low salt wash Buffer, 10 min at 4 °C on a rotator; 4 °C, 4,000 g, 2 min, discard supernatant.

(2) Preparing cell lysate (for 6-well plate)

1. Wash cells once with 0.5 ml of PBS.
2. 200 uL of Nondenatured Lysis Buffer per sample, resuspend cells and incubate 60 min at 4 °C on a rotator.
3. 4 °C, 12,000 rpm, 10 min, transfer supernatant into new 1.5 ml microfuge tube.
4. Add 1ug antibody to 200 ul lysate, at least 4 hours or overnight at 4 °C on a rotator.

(3) Preparing Co-IP (for 6-well plate)

1. Lysate (incubated antibody) add into protein A beads of blocked.
2. Incubate overnight at 4 °C on a rotator.

(4) Wash (for 6-well plate)

1. 4 °C, 4,000 g, 2 min, discard supernatant.
2. Wash beads 3 times(or twice) with 0.5 ml of Low salt wash Buffer, 10 min at 4 °C on a rotator; 4 °C, 4,000 g, 2 min, discard supernatant.
3. add 30 ul 1 x SDS elution buffer per sample, resuspend beads, 100 °C, 10 min.
4. Cool to room temperature, save in -80 °C.