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| ==Notes== | | ==Notes== |
| * 6-wells put to S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/23|23March2010]] were passed on to Anouk to continue --> VASP experiment cancelled due to time limitation | | * 6-wells put to S<sup>0</sup> [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/22|22March2010]] were passed on to Anouk to continue --> VASP experiment cancelled due to time limitation |
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| ==Results== | | ==Results== |
| *{{todo| WHAT?}} | | *{{todo| WHAT?}} |
Revision as of 01:39, 23 March 2010
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Project name
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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)
Notes
- 6-wells put to S0 22March2010 were passed on to Anouk to continue --> VASP experiment cancelled due to time limitation
Results
Conclusion
Related
Related entries
Same actions
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