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Summary
- Cell lysis Co-IP
- Co-IP Day II
- Stimulation
- Western blot, secondary antibody
- CHECK AVAILABILITY WESTERN BLOT THURSDAY!
- REGISTER FOR MLII 24/3/10
Materials & Methods
Materials
Method
- Use cells put on S0 23Feb2010
- Put cells on ice
- Remove medium
- Wash twice with 5 mL cold PBS
- Add 1 mL RIPA buffer
- Scrape of cells and collect in 1.5 mL tube
- Sonicate (4x short pulse)
- Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination)
Co-immunoprecipitation
- resuspend beads prepared 23Feb2010
- Prepare for all donors (D9 & D12)
- Only beads (30 μL)
- Beads w. antibody (30 μL)
- Beads w. antibody (30 μL) & lysis buffer (200 μL)
- Beads w. antibody (30 μL) & sample (200 μL)
(possible also beads (30 μL) with only sample (200 μL))
hTERT stimulation
- Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S0)
- Rinse twice with warm PBS and remove buffer
- Add 200 μL DMEM S0 with either HT31 or HT31P (see schedule below)
- Add 400 μL DMEM S0 to CTR (lane A)
- Add 200 μL DMEM S0 to CSE (lane B)
- Add 200 μL 8-pctp or 6-benz according to schedule below (lane C, D)
- Wait 25 min.
- Prepare 25 mL DMEM S0 with 100% CSE
- Dilute 100% CSE to 45% CSE
- Add 200 μL 45% CSE to wells according to schedule below (lane B, C D)
- Incubate 24 h.
Stimulation set-up
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S0
1
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S0
2
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S0
3
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HT31
4
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HT31
5
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HT31
6
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A
(CTR)
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B
(CSE)
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C
(CSE+8-pcpt)
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D
(CSE+6-Benz)
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CTR = control, CSE = Cigarette Smoke extract, 8-pcpt = 8-pcpt-2'-o-me-camp
Western blot, secondary Antibody
- Wash 3x 10 min. in TBST buffer (first can be less)
- Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT)
- Wash 3x 10 min. in TBST buffer
Preparing film
- 1:50 ratio ECL solutions B:A (2 mL per membrane)
- Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
- Place blot in between two overhead sheets
- Take picture (depends on what is available)
Results
Conclusion
Related
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