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
- Protein marker
- KAH127-7 (1/28/10, 24-well)
- KAH130-2 (2/3/10, 6-well)
- KAH130-7 (2/3/10, 6-well)
- KAH131-6 (2/3/10, 6-well)
- KAH131-9 (1/28/10, 24-well)
- KAH132-4 (1/28/10, 24-well)
- KAH132-6 (1/28/10, 24-well)
- KAH133-1 (1/28/10, 24-well)
- KAH133-2 (1/28/10, 24-well)
- KAH132-8 (1/25/10, 24-well) + ctrl
- KAH133-6 (1/25/10, 24-well) + ctrl
- Flp-in T-REx (parent line, no insert) - ctrl
- Protein marker
- 1x loading dye
|
Ponceau S stained filter
|
> Electroblot 2
--> Run @ 100 V for 1 hr., 15 min. (EED shows up at 80kD, according to Abcam)
Gel 1
- Protein marker
- KAH139/V0201, 0 neo
- KAH139/V0201, 50 μg/mL neo
- KAH139/V0201, 100 μg/mL neo
- KAH139/V0201, 200 μg/mL neo
- KAH139/V0201, 400 μg/mL neo
- KAH139/V0201, 500 μg/mL neo
- KAH140/V0201 (const. Gal4-EED)
- KAH141/V0201 (EED, no Gal4 DB)
- mock (no DNA in transfection)
- Protein marker
- 1x loading dye
- 1x loading dye
- 1x loading dye
- 1x loading dye
|
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
- Protein marker
- KAH127-7 = 43 kD
- KAH130-2 = 79 kD
- KAH130-7 = 79 kD
- KAH131-6 = 79.5 kD
- KAH131-9 = 79.5 kD
- KAH132-4 = 35 kD
- KAH132-6 = 35 kD
- KAH133-1 = 29.5 kD
- KAH133-2 = 29.5 kD
- KAH132-8 (+) = 35 kD
- KAH133-6 (+) = 29.5 kD
- Flp-in T-REx (parent line, no insert) - ctrl
- Protein marker
|
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
- Protein marker
- KAH139/V0201, 0 neo = 91 kD
- KAH139/V0201, 50 μg/mL neo = "
- KAH139/V0201, 100 μg/mL neo = "
- KAH139/V0201, 200 μg/mL neo = "
- KAH139/V0201, 400 μg/mL neo = "
- KAH139/V0201, 500 μg/mL neo = "
- KAH140/V0201 (const. Gal4-EED) = "
- KAH141/V0201 (EED, no Gal4 DB) = 74 kD
- mock (no DNA in transfection)
- Protein marker
GAPDH = 37 kD
|
15 min. exposure
|
--> No bands detected. Strip filter w/ 1x PBST (R.T./ overnight) and re-probe with higher conc. anti-EED (tomorrow)
|