User:Karmella Haynes/Notebook/Polycomb project/2010/01/25

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01/25/10

  • ✓ Polycomb-ATF lines: frozen stocks for confluent plates KAH
  • ✓ Cultures for Western blot: 12-well dish (~2.5x105/well) for cell lines above
  • ✓ Western blot: KAH126-1, -3, -4; KAH127-3, -4; KAH128-2, -6, -8; KAH129-3, -4, -8; KAH132-8; KAH133-6

Western blot
> Bradford assay
--> Calculate amount of protein to load in each lane (6 μL max)



> SDS-PAGE
--> Bring protein vol. up to 6μL w/ dH2O
--> Make 100 mM DTT in 2x loading buffer (200 μL total; > enough for 6 μL/ gel lane)
--> Add 6 μL DTT + buffer to each protein sample
--> Incubate at 100°C/ 5 min.
--> Gel loading:

Gel
  1. Protein marker
  2. KAH126-1
  3. KAH126-3
  4. KAH126-4
  5. KAH127-3
  6. KAH127-4
  7. KAH128-2
  8. KAH128-6
  9. KAH128-8
  10. KAH129-3
  11. KAH129-4
  12. KAH129-8
  13. KAH132-8
  14. KAH133-6
  15. Flp-in T-REx (parent line, no insert)
 1/25/10 Ponceau S stain
Ponceau S stained filter

> Electroblot
--> Run @ 100 V for 1 hr., 15 min. (for large protein transfer)


> Immunostaining
--> Primary: mouse anti-myc, 1:5000 (4°C/ overnight)
--> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.)

Results: Blot showed no signal in any lanes, except for a very faint signal in lane 14. Try again with max. volume of protein.


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