User:Karmella Haynes/Notebook/Polycomb project/2010/05/23

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05/23/10

  • ✓ Growth assay: cell count (day 6); split stock cultures (1x 10 cm plain medium, 1x 10 cm +antibiotics per line; 1:10)
  • ✓ Western blot: Pc-ATF negative ctrl lines


Western blot

> SDS-PAGE
--> Pc-ATF negative ctrl lines
--> 4x 10-well tris-glycine (4-20%) pre-cast gels
--> Samples (25 μL total vol./ well); use 15 μL PageRuler Plus pre-stained ladder
--> Make 100 mM DTT in 2x loading buffer (> enough for 12.5 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.

Gel 1
1. KAH154-1
2. KAH154-1 +dox
3. KAH154-2
4. KAH154-2 +dox
5. KAH154-4
6. KAH154-4 +dox
7. KAH155-2
8. KAH155-2 +dox
9. KAH155-5

Gel 2
10. KAH155-5 +dox
11. KAH155-6
12. KAH155-6 +dox
13. KAH156-2
14. KAH156-2 +dox
15. KAH156-3
16. KAH156-3 +dox
17. KAH156-5
18. KAH156-5 +dox

Gel 3
19. KAH157-1
20. KAH157-1 +dox
21. KAH157-2
22. KAH157-2 +dox
23. KAH157-5
24. KAH157-5 +dox
25. KAH158-2
26. KAH158-2 +dox
27. KAH158-3

Gel 4
28. KAH158-3 +dox
29. KAH158-4
30. KAH158-4 +dox
31. KAH159-1
32. KAH159-1 +dox
33. KAH159-2
34. KAH159-2 +dox
35. KAH159-3
36. KAH159-3 +dox

5/18/10 Ponceau S stain
5/18/10 Blot 4 re-done Ponceau S stain
Ponceau S stained filters

--> Blot #4 messed up, forgot to add top sponge to blot; Do over


> Immunostaining
--> Block 4°C/ overnight; continue Mon.

5/24/10
--> Primary (4°C/ overnight):

  1. rabbit anti-myc (ab1791), 1:4000, 10 mL

5/25/10
--> Secondary: donkey anti-rabbit IgG-HRP, 1:5000 (R.T./ 1 hr.) (Molecular weights computed at http://www.expasy.ch/tools/pi_tool.html)

Signals
  • KAH154-157 (1-24) = 37.5 kD
  • KAH158 (25-30) = 73.5 kD
  • KAH159 (31-36) = 74 kD
 Western blot 5/25/10

--> Oops! Mixed up a couple of samples:
33. 159-2
34. 159-3
35. 159-3 +dox
36. 159-2 +dox

> Conclusions: Use the following lines in further experiments

  1. KAH154-2 (hPcCD/-VP64)
  2. KAH155-2 (mPcCD/-VP64)
  3. KAH156-5 (fshPcCD/-VP64)
  4. KAH157-1 (flyPcCD/-VP64)
  5. KAH158-4 (hCBX8/-VP64)
  6. KAH159-2 (flyPc/-VP64)


Growth assay (day 6)
> Do cell counts for the following:

  1. KAH126-1
  2. KAH126-3
  3. KAH132-8
  4. FTRx
  5. KAH126-1 +dox
  6. KAH126-3 +dox
  7. KAH132-8 +dox
  8. FTRx +dox

--> Omitted KAH126-4; no RFP detected on day 4; lost RFP expression?
> Trypsinize cells with 200 μL Trypsin buffer
> Resuspend cells with 800 μL medium (1 mL total volume)
> Dilute samples 1/4 for Scepter counting; add 25 μL cells to 75 μL PBS


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