User:Karmella Haynes/Notebook/Polycomb project/2010/01/27

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01/27/10

  • ✓ Protein preps: dox-induced Pc-ATF lines from 12-well plate
  • ✓ Western blot: repeat from 1/25 using max. vol. protein

> 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 (from 1/23/10)
  15. Flp-in T-REx (parent line, no insert)
 1/27/10 Ponceau S stain
Ponceau S stained filter

> Electroblot
--> Run @ 100 V for 1 hr., 10 min. (for large protein transfer)


> Immunostaining
--> Primary: mouse anti-myc, 1:2500, 5 mL (4°C/ overnight)
--> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.)

Gel
  1. Protein marker
  2. KAH126-1 = 43 kD
  3. KAH126-3 = 43 kD
  4. KAH126-4 = 43 kD
  5. KAH127-3 = 43 kD
  6. KAH127-4 = 43 kD
  7. KAH128-2 = 43 kD
  8. KAH128-6 = 43 kD
  9. KAH128-8 = 43 kD
  10. KAH129-3 = 43 kD
  11. KAH129-4 = 43 kD
  12. KAH129-8 = 43 kD
  13. KAH132-8 = 35 kD
  14. KAH133-6 (from 1/23/10) = 29.5 kD
  15. Flp-in T-REx (parent line, no insert)
 Western blot 1/28/10
10 min. exposure



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