免疫共沉淀

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