Co-IP
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Protocol for Co-IP of different proteins with RFP-MeCP2
Preperation of cells (for p100):
• 1st day split 293T EBNA cells in DMEM (high glucose + 10% FCS + 5mM glutamine + 5µg/ml gentamycine)
• 2nd day cells should be 80-90% confluent
Transfection (TransFectin, Biorad):
• 15 µg plasmid DNA in 250µl serum-free DMEM (Eppi)
• 25 µl TransFectin in 250µl serum-free DMEM (Eppi)
• add TransFectin solution to DNA solution and mix by tapping or pipetting and incubate for 20min at RT
• add the DNA-TransFectin complex directly to the cells and swirl gently
• DMEM should be changed after 2h
• let cells grow over night
• wash 1x with 10ml PBS
• resuspend cells in 1.5ml PBS and transfer to an Eppy
• centrifuge for 3min at 9,000rpm, remove supernatant
• freeze pellets until further use
Co-IP:
1. resuspend pellets in 100µl EBC-buffer
2. take 20µl for Input control, + 7µl 4x sample buffer and boil 6min at 100°C put at -20°C
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 20µl RFP + 400µl 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 appropriate amount 4x sample buffer, boil 10min at 100°C put at -20°C
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 at 100°C put at -20°C
10. Next day: load samples to SDS-Page and proceed with WB
