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
2/5/10 Ponceau S stain
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
Image:KAH 020510 wb1.tif
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
Image:KAH 020510 wb2.tif
15 min. exposure



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