User:Karmella Haynes/Notebook/Polycomb project/2010/10/12

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10/12/10

  • ✓ ChIP qPCR: trouble shooting
  • ✓ Order oligos: new qPCR primer pairs
  • ✓ Western: ChIP/ co-IP optimization



ChIP qPCR troble shooting

  • Low C(t) values: primers too dilute; 750 nM = 30 μL of 10 μM primer mix in 400 μL total volume (not 500 μL total volume!)
  • GAPDH B is more efficient than experimental primers. Check GC content and Tm's of primers. Use Primer 3 to pick better primer pairs



Western: ChIP co-IP
> Optimization IP for αH3K27me3
> IP samples from 10/08/10; 60 μL each, ready to load
> Prep input samples: 22.5 protein + 7.5 4x loading dye
--> 4x loading dye (Invitrogen) w/ freshly added DTT (200 mM final)
> Use 10-well gel (loading volume = 25 μL)
> Electroblot: 1 hr. 30 min.

Gels 1 & 2

1. PageRuler Plus (10 μL)
2. KAH126-1 dx input
3. [KAH126-1 dx + α-H3K27me3] + ProtA seph beads
4. [KAH126-1 dx + α-IgG] + ProtA seph beads
5. [ProtA seph beads + α-H3K27me3] + KAH126-1 dx
6. [ProtA seph beads + α-IgG] + KAH126-1 dx
7-10. 1x dye

Image:KAH 101210 ps1.tif
Ponceau S stained filters

> Block: 5% milk/PBST, 4°C/ > 1 hr.

> Primary staining: 5% BSA/PBST, 4°C/o.n.

  1. rabbit α-DsRed 632496, 1:1000, 2.5 mL
  2. rabbit α-H3K27me3 07-449, 1:1000, 2.5 mL


10/13/10
> Secondary staining: 5% milk/PBST, R.T./ 1 hr.

  1. donkey α-rabbit-HRP, 1:5000; 5 mL

> Predicted sizes (using http://www.expasy.ch/tools/pi_tool.html for KAH proteins)

  • KAH126-1: 43 kD
  • H3K27me3: 15 - 17 kD


Image:KAH 101310 WB1.tif
Conclusions: IP is not specific to antibody. Next time, try ChIP protocol from Casey




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