User:Karmella Haynes/Notebook/Polycomb project/2010/01/21

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01/21/10

  • ✓ Western blot: Polycomb-ATF lines, check size of fusion protein w/ anti-myc

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
  1. Protein marker
  2. KAH126-1
  3. KAH126-3
  4. KAH126-4
  5. KAH127-3
  6. KAH127-4
  7. KAH127-7
  8. KAH128-2
  9. KAH128-6
  10. KAH128-8
  11. KAH129-3
  12. KAH129-4
  13. KAH129-8
  14. KAH126-7 no Dox (neg. ctrl)
  15. Protein marker
Gel #2
  1. Protein marker
  2. KAH130-2
  3. KAH130-7
  4. KAH131-1
  5. KAH131-6
  6. KAH131-9
  7. KAH132-4
  8. KAH132-6
  9. KAH132-8
  10. KAH133-1
  11. KAH133-2
  12. KAH133-6
  13. KAH126-7 no Dox (neg. ctrl)
  14. Protein marker
  15. 1x loading buffer


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



--> KAH133 (mCherry-only constructs) look good
--> Repeat this expt. for all others (seed 12-well plates for new protein preps)



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