12/01/10
- ✓ ChIP: pull-down; elute for DNA purification
ChIP co-IP optimization
> Samples:
- FTRx dx + α-H3K27me3 (07-449)
- FTRx dx + rabbit α-myc
- FTRx fx + α-H3K27me3 (07-449)
- FTRx fx + rabbit α-myc
> Buffers:
- Complete Buffer III
- Low Salt Immune Complex Buffer
- High Salt Wash Buffer
- LiCl Immune Complex Wash Buffer
> Bind beads (Note: *H3K27me3 ab 07-449 was Protein A purified, Millipore)
- Pellet Protein A* beads slurry, wash 3x with Complete Buffer III, and resuspend in original volume (store remainder at 4°C).
- Bind protein-ab complexes with 20 μL beads; rotate at 4°C/ 5 hours
Washes (Casey's protocol)
> Low Salt Immune Complex Buffer (LSIC) wash (2x, on ice)
- Pellet beads at 3000 rpm/ 4 °C/ 2 min.
(Note: beads+complexes can be kept on ice for 1-2 hours)
- Gently resuspend beads in 500 μL LSIC. Transfer to a fresh tube. Keep old tip inside original tube.
- Incubate beads/LSIC on ice/ 2 min. (flick once during incubation).
- Pellet beads at 3000 rpm/ RT/ 1 min.
- With a fresh tip, add 500 μL LSIC to old tube w/ old tip. Use old tip to transfer LSIC to pellet (to collect up all remaining beads)
- Repeat the previous wash and spin.
> High Salt Buffer (HSB) wash (1x, on ice)
- Gently resuspend beads in 1 mL HSB.
- Incubate beads/HSB on ice/ 2 min. (flick once during incubation).
- Pellet beads at 3000 rpm/ RT/ 1 min.
> LiCl Immune Complex Buffer (LICB) wash (1x on ice)
- Gently resuspend beads in 1 mL LICB.
- Incubate beads/LICB on ice/ 2 min. (flick once during incubation).
- Pellet beads at 3000 rpm/ RT/ 1 min.
> TE buffer wash (1x, RT)
- Gently resuspend beads in 1 mL TE pH 7.5.
- Incubate beads/TE @ RT/ 2 min. (flick once during incubation).
- Pellet beads at 3000 rpm/ RT/ 30 sec.
Elution (Qingqing's protocol)
> Elute for DNA purification
- Resuspend beads in 500 ul 1% SDS/TE buffer.
- Heat at 65°C/ 15 min., vortex every 2 min.
- Pellet beads, at 5000 rpm/ RT/ 3 min.
- Transfer eluted chromatin (sup.) to a new 1.5 ml tube
DNA prep: day 1 (Qingqing's protocol)
- Thaw 100 μL input sample. Add 400 μL 1% SDS/TE.
- Add 20 μL Pronase (20 μg) to each ChIP and Input sample.
- Incubate at 42°C/ 1 hr.
- Incubate at 65°C/ 48 hrs
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