简单的步骤！

Co-IP：

1.resuspend pellets in 100µl EBC-buffer

2.take 20µl for Input control，+ 7µl put at -20℃?4x sample buffer and boil 6min at 100℃

3.add 400µl EBC-buffer and incubate for 1h at 4?C in 360? shaker

4.put 100µl Immobilized rProtein A in two separate Eppies and equilibrate 3x with 500µl EBC-buffer （i.e.add EBC buffer，mix gently，spin down and remove supernatant）

a.1x for antibody binding： add 20&RFP + 400?micro；l µl EBC-buffer to equilibrated beads and incubate for 1h at 4?C in 360? shaker

b.1x for preclearing of extracts： add extract （from step 3）to equilibrated beads and incubate for 1h at 4?C in 360? shaker

5.wash beads for antibody binding （from step 4a）3x with 500µl EBC-buffer and add precleared extract to beads （from step 4b）

6.incubate for 2.5h at 4?C in 360? shaker

7.spin down beads，keep supernatant （flow through； ~500µl）and add put at -20℃?appropriate amount 4x sample buffer，boil 10min at 100℃

8.wash 3x with 1ml EBC-buffer

9.resuspend beads in 100µl EBC-buffer and add 35µl 4x sample buffer （bound fraction）or to load total amount of bound fraction，add 30µl 1x sample buffer，boil 10min put at -20℃?at 100℃

10.Next day： load samples to SDS-Page and proceed with WB