IP - Immunoprecipitation

This technique is used for isolating proteins out of a solution using an antigen-antibody interaction, which are then pulled out of the samples by protein A/G-coupled agarose beads or magnetic beads.

Required Material:

• PBS

• Cell lysis buffer

• SDS buffer

• Low -pH Glycine buffer

• Refrigerated microcentrifuge

• Sonicator

• Protein A/G agarose beads

• Antibody

Protocol:

Preparation of Cell Lysates:

1. Spin the collected cells at 500xg for 5 minutes at 4°C and then remove the supernatant.

2. Wash the cells with PBS 3 times, spin again at 500xg for 5 minutes and remove the supernatant.

3. Resuspend the washed cell pellet in cell lysis buffer (1ml per 1x10⁷cells) and incubate on ice for 10 minutes.

4. Sonicate the cells three times for 5 seconds to ensure full release of the proteins from cells.

5. Spin the pellet cellular debris at 13,000xg for 10 minutes and transfer the supernatant into a new tube. The supernatant can be stored for long-term at -80°C for future use.

Preparation of Protein A/G agarose beads:

1. A/G agarose beads need to be washed with 1mm PBS and centrifuged at 2,000xg for 30 seconds. Remove the collected supernatant and repeat the wash.

2. Dilute beads 1:1 with PBS.

Note: When manipulating agarose beads it is recommended to use wide pipette tips or tips with the end cut off to avoid the beads getting disrupted.

Immunoprecipitation:

1. (Optional) Pre-clear the cell lysate: add 100µl pre-washed Protein A/G agarose bead slurry per 1 mL of cell lysate and incubate at 4°C for 10 min with gentle agitation. Spin at 3,000×g at 4°C for 2 min, remove the beads and collect the supernatant in a new centrifuge tube.

2. Add the antibody to the pre-cleared cell lysate and gently rock the mixture at 4°C for 2 - 4 hours.

3. To capture the immunocomplex add 25 - 40µl of pre-washed Protein A/G agarose bead slurry and gently mix for 1 hour on a rocking platform.

4. Centrifuge the mixture at 10,000xg for 30 seconds at 4°C.

5. Carefully remove all the supernatant and wash the beads 3-5 times with 500µl of Lysis buffer.

Elution:

The protein can be eluted from the beads by SDS buffer (denaturing) or Low-pH glycine buffer (non-denaturing).