User:Karmella Haynes/Notebook/PcTF Genomics/2015/08/13

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08/13/15

  • Western Castone - blocking & primary staining

Western Castone - blocking & primary staining

Imaging

  • Got Pxi4 imager working!
  • Recorded Ponceau stained membranes (were stored in di-water at 4°C overnight)


Blocking

  • Blocked in 5% dry milk/ 1xPBST ~1 hour/ room temp on orbital platform (in petri dishes)
  • Ponceau stain was completely removed


Primary Stain

  • Diluted anti-H3K27me3 07-449 (rabbit poly) 1:500 in 5% dry milk/ 1xPBST
  • Used "floating" method:
    • cut 2x parafilm squares to fit size of membrane
    • stacked on top of each other and pressed onto bottom of 10 cm petri dish
    • applied 500 μL diluted primary near (but not at) bottom of parafilm square
    • carefully laid membrane face-down onto antibody sln.
    • applied petri lid and sealed with parafilm
    • Incubated @ 4°C (cold room) on shelf overnight


Trouble-shooting note: May want to switch to BSA-based blocking solution. Had trouble with dry milk before at Harvard. See Abcam notes, http://www.abcam.com/protocols/histone-western-blot-protocol



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