- Western Castone - blocking & primary staining
Western Castone - blocking & primary staining
- Got Pxi4 imager working!
- Recorded Ponceau stained membranes (were stored in di-water at 4°C overnight)
- Blocked in 5% dry milk/ 1xPBST ~1 hour/ room temp on orbital platform (in petri dishes)
- Ponceau stain was completely removed
- 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