磁珠法免疫共沉淀（Co-IP）详细操作Protocol（英文

This protocol offers a general guideline for immunoprecipitation. Optimization may be required for each antibody and target antigen. The protocol uses 25 μL of Magnetic Beads, but this may be scaled up or down as required.

For each sample, you will need 25 μL of magnetic beads for preclearing and IP reaction. Prepare the beads immediately before use.

1. Transfer the required amount of beads to a clean microcentrifuge tube with 200 μL IP buffer.

2. Place the tubes containing beads on a magnetic rack for 1 minute. Carefully discard the supernatant.

3. Remove the tubes from the magnetic rack. Resuspend the beads in 200 μL of IP buffer by pipetting gently up and down. Incubate at RT for 10S.

4. Place the tubes on a magnetic rack for 1 minute and discard the supernatant.

5. Repeat the wash steps 1.3-1.4 two more times. Resuspend the beads in 200 μL IP buffer. Do not allow the beads to dry out.

1. Lyse the cells (around 1×10⁷) in 400 μL IP buffer (supplemented with 1×proteinase inhibitor cocktail and 1mM PMSF) by incubating on ice for 30 min, spin down cell debris and nuclei by centrifugation at 10,000g for 10 minutes at 4℃. (Save 10 μL supernatant in 10 μL 2× SDS sample buffer and boiled at 100 ℃ for 10 minutes. )

2. Preclear the lysate by combing 25 μL of magnetic beads with above sample lysate in a microcentrifuge tube. Incubate the tube at 4 ℃ with gentle rotation for 30 min.

3. Place tubes on a magnetic rack for 1 minute and transfer the supernatant into a new microcentrifuge tube placed on ice.

4. To the precleared lysate, add appropriate amount of primary antibody (0.5 μg～1.0 μg ). Incubate the sample at 4 ℃ for 2 hours with gentle rotation.

5. Place the tubes containing magnetic beads (prepared in steps 1.1-1.5) on a magnetic rack for 1 minute and discard the supernatant. Add magnetic beads to the samples. Incubate the samples at room temperature for 1 hour with gentle rotation.

6. Place the tubes on a magnetic rack for 1 minute. Discard the supernatant.

7. Remove the tubes from the magnetic rack, and resuspend the beads in 200 μL IP buffer by pipetting gently up and down.Incubate the tubes on ice for 1 minute.

8. Place the tubes on a magnetic rack for 30 sec. Discard the supernatant.

9. Repeat the wash steps 2.7-2.8 two more times.

10. Add 25μL of 2× SDS sample buffer to the beads. Boiled the samples at 100 ℃ for 5 minutes.

11. Place the tubes on a magnetic rack for 1 minute.

12. Load the Boiled samples onto the wells of SDS-PAGE gel.

Equipment:

Solution rotator, magnetic rack

IP buffer:

50 mM Tris-HCl (PH 7.4)

150 mM NaCl

2 mM EDTA

1% NP-40


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