02/18/10
- ✓ Western: Polycomb-ATF lines (new 10-well gel)
Western blot
> SDS-PAGE
--> Use new 10-well tris-glycine (4-20%) pre-cast gel
--> Samples (25 μL total vol.):
- Pre-stained ladder: 20 μL
- 126-1 - 132-8, FTRx: 12.5 μL protein
- 133-1: 6.25 μL protein (+6.25 dH2O)
--> Make 100 mM DTT in 2x loading buffer (200 μL total; > enough for 6 μL/ gel lane)
--> Add 12.5 μL DTT + buffer to each protein sample
--> Incubate at 100°C/ 5 min.
--> Run gel @ 120 V until dye front reaches the bottom
> Electroblot
--> Run @ 100 V for 1 hr., 30 min.
Gel loading
- BenchMark pre-stained ladder
- KAH126-1
- KAH127-4
- KAH128-8
- KAH129-4
- KAH130-2
- KAH131-9
- KAH132-8
- KAH133-1
- FTRx (parent line)
|
Ponceau S stained filter
|
> Immunostaining #1
--> Primary: mouse anti-myc tag, 1:5000, 10 mL (4°C/ overnight)
--> Secondary: donkey anti-mouse-HRP, 1:10,000 (R.T./ 1 hr.)
--> RESULT: Blank. Strip filter in 1x PBST overnight and re-probe w/ anti-DsRed
> Immunostaining #2 (2/22/10)
--> Primary: rabbit anti-DsRed, 1:4000, 10 mL (4°C/ overnight)
--> Secondary: donkey anti-rabbit IgG-HRP, 1:5,000 (R.T./ 1 hr.)
(Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)
Pc-ATF lines
- Protein marker
- KAH126-1 = 43 kD
- KAH127-4 = 43
- KAH128-8 = 43
- KAH129-4 = 43
- KAH130-2 = 79
- KAH131-9 = 79.5
- KAH132-8 = 35
- KAH133-1 = 29.5
- U2OS Flp-in T-REx (parent)
|
15 min. exposure
|
--> Looks great. 131-9 not expressed very well
--> Strip o/n and re-probe w/ 1:1000 anti-myc
> Immunostaining #3 (2/24/10)
--> Primary: mouse anti-myc tag, 1:1000, 10 mL (4°C/ overnight)
--> Secondary: donkey anti-mouse-HRP, 1:5,000 (R.T./ 1 hr.)
Pc-ATF lines
- Protein marker
- KAH126-1 = 43 kD
- KAH127-4 = 43
- KAH128-8 = 43
- KAH129-4 = 43
- KAH130-2 = 79
- KAH131-9 = 79.5
- KAH132-8 = 35
- KAH133-1 = 29.5
- U2OS Flp-in T-REx (parent)
|
15 min. exposure
|
--> Non-specific signal
| 
Project name
Main project page
Previous entry Next entry
