Six Tips to Improve Your Co-IP Results

The following six tips will help you achieve successful co-IP results with SureBeads Magnetic Beads.

1. Samples

• Select biologically relevant samples that have your target protein complex
• Avoid high concentrations, harsh detergents like SDS, and additives that will cause protein complex dissociation

2. Immunoprecipitation

• Maintain protein complexes by using freshly prepared lysates
• Select capture reagent based on the host species of the capture antibody. Protein A has higher affinity for rabbit IgGs, while Protein G has higher affinity for mouse IgGs

3. Unidirectional Co-IP

• Due to differences in abundance, you may observe co-IP in one direction, rather than two. When this occurs, immunoprecipitate the component that has the highest percentage of its total population bound to its partner

4. Other Antibodies

• Try another antibody if you do not observe co-IP. Only unbound protein will immunoprecipitate if the epitope of the capture antibody overlaps the protein-protein interaction site

5. Positive and Negative Controls

• To show that the IP worked, include a blot for just the immunoprecipitated component (positive control)
• To show that the co-IP worked, include a blot for just the SureBeads Magnetic Beads or IgGs (negative control)

6. Analysis

• To avoid antibody detection in the western blot, use a detection antibody from a different host species than the antibody used in the IP

The following results were achieved using these tips and following the protocol as outlined:

Successful IP and Co-IP results. HEK293 whole cell lysates were immunoprecipitated with cdc2 using the SureBeads IP protocol. Cyclin B1 was successfully co-immunoprecipitated with cdc2.

Protocol: Co-IP with SureBeads Magnetic Beads

Sample Preparation

1. Determine the best cell line for target of interest.
2. Grow at least 3.6 x 10⁷ cells (two 80–95% confluent T-175 flasks).
3. Split cultures 24–48 hr prior to harvest.
4. Grow cells to mid-log growth (determine corresponding confluency or density).
5. Lyse cells using this lysis buffer:
   • 20 mM Tris (pH 7.5)
   • 150 mM NaCl
   • 1 mM EDTA
   • 1 mM EGTA
   • 1% Triton X-100
   • Add fresh protease inhibitor before use
6. IP freshly prepared lysates to avoid complex dissociation.
7. IP using SureBeads Magnetic Beads.

Co-Immunoprecipitation

1. Select SureBeads Protein A or Protein G Magnetic Beads appropriate for the antibody used for IP.
2. Vortex SureBeads to resuspend them and transfer 100 µl (1 mg at 10 mg/ml) of SureBeads to tube.
3. Magnetize beads and discard supernatant.
4. Wash 3x with 1 ml PBS-T (1x PBS + 0.1% Tween-20).
5. Add 7.5 µg of antibody. Rotate 20 min at room temperature.
6. Wash 3x with 1 ml PBS-T.
7. Add 1 mg of antigen-containing lysate. Rotate 1 hr at room temperature.
8. Magnetize beads and wash 3x with 1 ml PBS-T.
9. Elute with 20 µl of 2x Laemmli Buffer. Incubate for 5 min at 90°C.
10. Magnetize and transfer supernatant to new vial (IP sample).
11. Elute again with 20 µl of 2x Laemmli Buffer. Incubate for 5 min at 90°C.
12. Magnetize and transfer supernatant to new vial (IP sample).
13. Run SDS-PAGE.

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