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        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

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