User:Karmella Haynes/Notebook/Polycomb project/2010/11/02

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11/02/10

  • ✓ ChIP/ co-IP: bind beads, wash, elute
  • ✓ Change buffer for HepG2 transfections



ChIP co-IP optimization
> Try gentler wash with more aggressive elution to increase pull-down

> Samples (2 each):

  1. KAH126-1 fx + α-H3K27me3 (07-449)
  2. KAH126-1 fx + α-rabbit IgG
  3. KAH132-8 fx + α-H3K27me3 (07-449)
  4. KAH132-8 fx + α-rabbit IgG
  5. KAH130-4 fx + α-H3K27me3 (07-449)
  6. KAH130-4 fx + α-rabbit IgG


> Make buffers:

  1. Complete Buffer III
  2. Low Salt Immune Complex Buffer
  3. High Salt Wash Buffer
  4. 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 10 μL beads; rotate at 4°C/ 2 hours


> Low Salt Immune Complex Buffer (LSIC) wash (2x, on ice)

  • Pellet beads at 3000 rpm/ 4 °C/ 3 min.

(Note: beads+complexes can be kept on ice for 1-2 hours)

  • Resuspend beads in 500 μL LSIC. Transfer to a fresh tube. Keep old tip inside original tube.
  • Incubate beads/LSIC on ice/ 5 min. (flick twice during incubation).
  • Pellet beads at 3000 rpm/ RT/ 30 sec. Keep 100 μL supernatant "W1" for Western, discard the rest.
  • 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)

  • Resuspend beads in 1 mL HSB.
  • Incubate beads/HSB on ice/ 5 min. (flick twice during incubation).
  • Pellet beads at 3000 rpm/ 4 °C/ 3 min.


> LiCl Immune Complex Buffer (LICB) wash (1x on ice)

  • Resuspend beads in 1 mL LICB.
  • Incubate beads/LICB on ice/ 5 min. (flick twice during incubation).
  • Pellet beads at 3000 rpm/ 4 °C/ 3 min.


> TE buffer wash (1x, RT)

  • Resuspend beads in 1 mL TE pH 8.0.
  • Incubate beads/TE @ RT/ 5 min. (flick twice during incubation).
  • Pellet beads at 3000 rpm/ RT/ 3 min.


> Elute for Western

  • Elute with 30 μL 1x loading dye + 50 mM DDT (enough to load two Western wells)
  • Vortex, heat at 100°C/ 5 min., vortex.
  • Pellet beads at 4000 rpm/ RT/ 3 min., transfer supernatant to new tube
  • Store at -20°C until Western blot. (for Western, heat to 100°C/ 5 min. before loading)


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