Anti-Flag or HA or Myc Affinity Resin

• Centrifuge cell suspension at 1000 ×g for 5 minutes to pellet the cells. Discard the supernatant.
• Wash cells once by suspending the cell pellet in PBS. Centrifuge at 1000 ×g for 5 minutes to pellet cells.
• Add ice-cold Cell Lysis Buffer to the cell pellet. Use 500 μL of Cell Lysis Buffer per 50 mg of cell pellet (i.e., 10:1 v/w).
• Incubate cell lysate on ice for 5 minutes with periodic pipetting. Remove cell debris by centrifugation at 10,000 ×g for 5 minutes. Transfer supernatant to a new tube for protein concentration determination.

Co-IP

• Gently swirl the bottle of affinity resin to obtain an even suspension. Aspirate 20 μL of the resin slurry into a new Eppendorf tube.
• Centrifuge resin at 3,000 xg for 1min. Wash the resin with 200 μl of Resin Coupling Buffer and centrifuge again.
• Chill resin pellet on ice or proceed to Co-IP reaction.
• Aliquot the cell lysate into 100 μl volume. Each volume will be used for one antibody pulldown. Negative control antibody is an antibody that pulls down an irrelevant protein not participating the bait-prey interaction.
• Add the 100 μl volume of cell lysate to the pre-chilled affinity resin pellet.
• Incubate the lysate-resin solution on a rotator at room temperature for 30 minutes, ensuring that the slurry remains suspended during incubation.
• Centrifuge resin at 3,000 xg for 1min. Wash the resin with 200 μl of Co-IP Wash Buffer and centrifuge again. Repeat this step two more times.
• Resuspend the antibody-resin pellet with 100 μl of SDS Loading Buffer.
• Heat at 95^oC for 5min. Chill on ice. Centrifuge resin at 3,000 xg for 1min.
• Collect the supernatant, which will be ready for western blot.

Buffers in this protocol are from BiCell Scientific’s Co-Immunoprecipitation (Co-IP) Kit (Cat No: BCIPKT).

For Research Use Only. Not for use in clinical diagnostics.