User:Karmella Haynes/Notebook/Polycomb project/2010/02/05

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02/05/10

  • ✓ HEK293 #4 Luc: expand to 74 cm2 flask (non selective 10% FBS, 1% P/S DMEM)
  • ✓ Polycomb-ATF lines: thaw cells
  • ✓ Western: Gal4-EED set (same as 2/03/20); re-probe stripped filter w/ anti-EED; new blot for anti-Gal4



Thaw Polycomb-ATF lines
--> Pellet thawed cells in 4 ml 100% FBS
--> Plate cells in 20% FBS, 1% P/S McCoy's 5A

  1. KAH126-1 (human Pc-CD)
  2. KAH127-4 (mouse Pc-CD)
  3. KAH128-8 (fly Pc-CD)
  4. KAH129-4 (fish Pc-CD)
  5. KAH130-2 (human CBX8)
  6. KAH131-9 (fly Pc)
  7. KAH132-8 mCh-VP64
  8. KAH133-1 mCh



Western blot


> Stripped blot #2 (from 2/04)
--> Incubate in blocking solution @ R.T./ > 1 hr.


> SDS-PAGE
--> Make new blot of Gal4 staining --> Use 6μL protein for all samples
--> 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.
--> Run gel @ 120 V


> Electroblot
--> Run @ 100 V for 1 hr., 15 min.

Gel loading
  1. Protein marker
  2. KAH139/V0201, 0 neo
  3. KAH139/V0201, 50 μg/mL neo
  4. KAH139/V0201, 100 μg/mL neo
  5. KAH139/V0201, 200 μg/mL neo
  6. KAH139/V0201, 400 μg/mL neo
  7. KAH139/V0201, 500 μg/mL neo
  8. KAH140/V0201 (const. Gal4-EED)
  9. KAH141/V0201 (EED, no Gal4 DB)
  10. mock (no DNA in transfection)
  11. Protein marker
  12. 1x loading dye
  13. 1x loading dye
  14. 1x loading dye
  15. 1x loading dye
File:KAH 020510 ps1.tif
Ponceau S stained filter


> Immunostaining, Blot 2
--> Primary: mouse anti-EED, 1:1000, 5 mL (4°C/ overnight)
--> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)

Blot 2: Gal4-EED transients
  1. Protein marker
  2. KAH139/V0201, 0 neo = 91 kD
  3. KAH139/V0201, 50 μg/mL neo = "
  4. KAH139/V0201, 100 μg/mL neo = "
  5. KAH139/V0201, 200 μg/mL neo = "
  6. KAH139/V0201, 400 μg/mL neo = "
  7. KAH139/V0201, 500 μg/mL neo = "
  8. KAH140/V0201 (const. Gal4-EED) = "
  9. KAH141/V0201 (EED, no Gal4 DB) = 74 kD
  10. mock (no DNA in transfection)
  11. Protein marker
Western blot 2/3/10
15 min. exposure


> Immunostaining, Blot 3
--> Primary: mouse anti-Gal4, 1:5000, 5 mL (4°C/ overnight)
--> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)

Blot 2: Gal4-EED transients
  1. Protein marker
  2. KAH139/V0201, 0 neo = 91 kD
  3. KAH139/V0201, 50 μg/mL neo = "
  4. KAH139/V0201, 100 μg/mL neo = "
  5. KAH139/V0201, 200 μg/mL neo = "
  6. KAH139/V0201, 400 μg/mL neo = "
  7. KAH139/V0201, 500 μg/mL neo = "
  8. KAH140/V0201 (const. Gal4-EED) = "
  9. KAH141/V0201 (EED, no Gal4 DB) = 74 kD
  10. mock (no DNA in transfection)
  11. Protein marker
Western blot 2/3/10
15 min. exposure