User:Karmella Haynes/Notebook/Polycomb project/2010/10/06

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

10/06/10

  • ✓ Order oligos: new primers for MMP12 and TNF ChIP-qPCR
  • ✓ Cell culture: split HEK Gal4-EED's; set up dox-induced Gal4-EED time course plate (12-well, ~50,000 cells/well)
  • ✓ ChIP/ co-IP: Protein A Sepahrose-H3K27me3 IP



Oligos

  1. MMP12 C f2; gaattgcaagcagtggtgctg
  2. MMP12 C r2; cagaccgtccccatacaatc
  3. MMP12 C f3; gcagattcttgctgatctgttc
  4. MMP12 C r3; ctcacagcagaataaccagc
  5. MMP12 D f1; cataacagtccttcaaaacc
  6. MMP12 D r1; tggtctaggaggctgtctgc
  7. MMP12 D f2; ctgagatgactgagggtaaag
  8. MMP12 D r2; ggagttcacttctgttatgg
  9. TNF C f2; gagaagcaactacagacc
  10. TNF C r2; cgtgggtcagtatgtgagag
  11. TNF C f3; ccagatgagctcatgggtttc
  12. TNF C r3; gctgggtgtgccaacaactg
  13. TNF D f1; gtgaaagatgtgcgctgatag
  14. TNF D r1; cagacatcctgtctctccatc
  15. TNF D f2; catggaaggtgctcactaag
  16. TNF D r2; ccacatctgtctccatatc



ChIP co-IP optimization
> Not much luck with endogenous protein IP using Qingqing's protocol
> Try simplified approach:
--> Bind antibody with chromatin overnight
--> Bind Protein A* beads with complexes for 5 hours
Note: *H3K27me3 ab 07-449 was Protein A purified (Millipore)
--> Wash with complete sonication buffer (same as IP using myc-conjugated beads)
--> Elute with Western loading buffer


Today:
> Try...

  • chromatin + antibody, then beads and
  • beads + antibody, then chromatin

> Prepare complete sonication buffer (see ###)
> Prep 200 μL Protein A sepharose by washing and resuspending (3 times) in complete sonication buffer
> Antibody binding
--> Use H3K27me3 07-449
--> Use double x-linked KAH126-1 (surplus chromatin, not important)

Reagent 1 2 3 4
α-H3K27me3 07-449 2.0 --- 2.0 ---
rabbit IgG ab --- 2.0 --- 2.0
KAH126-1 dx 250.0 250.0 --- ---
Protein A beads --- --- 10.0 10.0
Sonication buffer 250.0 250.0 500.0 500.0

--> Rotate at 4°C/ overnight