User:Karmella Haynes/Notebook/Polycomb project/2010/02/03

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02/03/10

  • ✓ Protein prep: Gal4-EED transfections (from 2/02)
  • ✓ Western blot: 1. Polycomb-ATF lines, 2. Gal4-EED transfections
  • ✓ HEK293 #4 Luc cells: seeded culture from 2/20 not growing well; change to 20% FBS DMEM; thaw new tube, pellet in FBS, plate in 20% FBS DMEM


Western blot


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

Gel 1
  1. Protein marker
  2. KAH127-7 (1/28/10, 24-well)
  3. KAH130-2 (2/3/10, 6-well)
  4. KAH130-7 (2/3/10, 6-well)
  5. KAH131-6 (2/3/10, 6-well)
  6. KAH131-9 (1/28/10, 24-well)
  7. KAH132-4 (1/28/10, 24-well)
  8. KAH132-6 (1/28/10, 24-well)
  9. KAH133-1 (1/28/10, 24-well)
  10. KAH133-2 (1/28/10, 24-well)
  11. KAH132-8 (1/25/10, 24-well) + ctrl
  12. KAH133-6 (1/25/10, 24-well) + ctrl
  13. Flp-in T-REx (parent line, no insert) - ctrl
  14. Protein marker
  15. 1x loading dye
 2/3/10 Ponceau S stain
Ponceau S stained filter

> Electroblot 2
--> Run @ 100 V for 1 hr., 15 min. (EED shows up at 80kD, according to Abcam)

Gel 1
  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
 2/3/10 Ponceau S stain
Ponceau S stained filter


> Immunostaining 1
--> Primary: mouse anti-myc, 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 1: Polycomb-ATF lines
  1. Protein marker
  2. KAH127-7 = 43 kD
  3. KAH130-2 = 79 kD
  4. KAH130-7 = 79 kD
  5. KAH131-6 = 79.5 kD
  6. KAH131-9 = 79.5 kD
  7. KAH132-4 = 35 kD
  8. KAH132-6 = 35 kD
  9. KAH133-1 = 29.5 kD
  10. KAH133-2 = 29.5 kD
  11. KAH132-8 (+) = 35 kD
  12. KAH133-6 (+) = 29.5 kD
  13. Flp-in T-REx (parent line, no insert) - ctrl
  14. Protein marker
 Western blot 2/3/10
15 min. exposure

--> KAH130-2, KAH130-7, KAH131-6 are better, cleaner preps using more cells, but band undetectable. Dox induction showed very few RFP+ cells for 130-2 and none for 130-7 and 131-6
--> Repeat Western blot analysis after thawing and expanding select lines


> Immunostaining 2
--> Primary: mouse anti-EED, 1:5000, 5 mL (4°C/ overnight)
--> Secondary:

  • donkey anti-mouse-HRP, 1:10,000
  • anti-GAPDH-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

GAPDH = 37 kD

Western blot 2/3/10
15 min. exposure

--> No bands detected. Strip filter w/ 1x PBST (R.T./ overnight) and re-probe with higher conc. anti-EED (tomorrow)



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