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
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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
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Ponceau S stained filters
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--> 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):
- 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
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--> 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
- KAH154-2 (hPcCD/-VP64)
- KAH155-2 (mPcCD/-VP64)
- KAH156-5 (fshPcCD/-VP64)
- KAH157-1 (flyPcCD/-VP64)
- KAH158-4 (hCBX8/-VP64)
- KAH159-2 (flyPc/-VP64)
Growth assay (day 6)
> Do cell counts for the following:
- KAH126-1
- KAH126-3
- KAH132-8
- FTRx
- KAH126-1 +dox
- KAH126-3 +dox
- KAH132-8 +dox
- 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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