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Co-immunoprecipitation part II, (Stimulation hTERT cells v2)
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Summary
Conditions
- CTR
- CSE
- CSE + 8-pcpt
- CSE + 6-Bzn
- 8-pcpt
- 6-Bnz
With a dose response range of 0 - 50 μg/mLug Ht31, instead of HT31P S0 was used.
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))
Stimulation hTERT (BSc students
- HT31P dose response curve (0 - 50 μM)
- HT31 (200 μM) /S0 Basal
Results
Standard curve Pierce Assay
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Prot. Conc. (μg/mL)
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Av. Absorb. (595 nm)
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Intersect
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Slope
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R2
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A
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2000
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0.698
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0.1106
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0.0003
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0.993
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B
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1500
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0.527
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C
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1000
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0.421
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D
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750
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0.357
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E
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500
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0.256
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F
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250
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0.182
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G
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125
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0.153
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H
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0
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0.090
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Measurement concentration samples
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Av. Absorb. (595 nm)
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Prot. Conc. (mg/mL)
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Prot. Conc. (corrected)(mg/mL)
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D9
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0.542
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1.470
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D9 (1:5)
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0.219
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0.368
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1.84
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D12
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0.541
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1.466
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D12 (1:5)
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0.204
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0.317
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1.58
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Related entries
Run 2: Ht31 Dose/Effect Curve & S0 D9/D12
Same actions
Related topics
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