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
File:KAH 020310 ps1.tif
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
File:KAH 020310 ps2.tif
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
File:KAH 020310 wb1.tif
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

File:KAH 020310 wb2.tif
15 min. exposure

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