Transfect cells…
- collect after 1 day. - Remove medium
- Wash cells off plate in 10 mL cold PBS - Spin down gently (approx 5 min @ 2k rpm) - Remove PBS
- Bring up cells in 300 ul polysome lysis buffer (PEB) - Lyse on ice, 5 min - Freeze at –80c
Prepare antibody-coated beads (1 day ahead)…
- Protein A agarose beads (Sigma P3391). Amount? Swell in ?? NT2 buffer
o Add 5% BSA
- 10 ul anti-GFP antibody per reaction (Sigma G1544) - rotate end-over, cold, 1 hr to overnight
o can be stored at 4 degrees if azide (.05%) is added. (up to 6 months)
immunoprecipitation: - thaw lysate on ice
- centrifuge 14,000 g for 15 minutes, cold - remove sup, store on ice until use
o store extra at –80c -
- wash coated beads 4 – 6x in NT2 - resuspend in 850 ul NT2 -
- set up reaction:
100 ul mRNP lysate 10 ul DTT (100mM) 30 ul EDTA (0.5M)
5 ul RNase Inhibitor (NEB) 1 mL total
- mix, quickly remove 100 ul to represent total RNA - mix both end-over at RT, 2 hrs. or O/N, cold.
- Pulse in centrifuge to pellet beads - wash 4-6x with 1 mL NT2 buffer
- resuspend in 100 ul NT2 and 100 ul SDS-TE
o incubate 30 min @ 55 C.
o remove sup from beads to new tube. -
- isolate RNA from all samples (total, IP) using TRIZOL LS
o check quality on nanodrop using 260/280 AND 260/230. both should be near above 1.6, near 2.0.
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