User:Karmella Haynes/Notebook/Polycomb project/2010/02/18

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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
  1. BenchMark pre-stained ladder
  2. KAH126-1
  3. KAH127-4
  4. KAH128-8
  5. KAH129-4
  6. KAH130-2
  7. KAH131-9
  8. KAH132-8
  9. KAH133-1
  10. FTRx (parent line)
2/5/10 Ponceau S stain
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
  1. Protein marker
  2. KAH126-1 = 43 kD
  3. KAH127-4 = 43
  4. KAH128-8 = 43
  5. KAH129-4 = 43
  6. KAH130-2 = 79
  7. KAH131-9 = 79.5
  8. KAH132-8 = 35
  9. KAH133-1 = 29.5
  10. U2OS Flp-in T-REx (parent)
Western blot 2/22/10
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
  1. Protein marker
  2. KAH126-1 = 43 kD
  3. KAH127-4 = 43
  4. KAH128-8 = 43
  5. KAH129-4 = 43
  6. KAH130-2 = 79
  7. KAH131-9 = 79.5
  8. KAH132-8 = 35
  9. KAH133-1 = 29.5
  10. U2OS Flp-in T-REx (parent)
Western blot 2/24/10
15 min. exposure

--> Non-specific signal



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