User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/24

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Co-immunoprecipitation part II, (Stimulation hTERT cells v2) Main project page
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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

Determination of protein concentration

  • 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)
  1. Only beads (30 μL)
  2. Beads w. antibody (30 μL)
  3. Beads w. antibody (30 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (30 μL) & sample (200 μL)

(possible also beads (30 μL) with only sample (200 μL))

  • Incubate ON @ 4 °C

Stimulation hTERT (BSc students

  • HT31P dose response curve (0 - 50 μM)
  • HT31 (200 μM) /S0 Basal

Results

Standard curve Pierce Assay
Prot. Conc. (μg/mL) Av. Absorb. (595 nm) Intersect Slope R2
A 2000 0.698 0.1106 0.0003 0.993
B 1500 0.527
C 1000 0.421
D 750 0.357
E 500 0.256
F 250 0.182
G 125 0.153
H 0 0.090



Measurement concentration samples
Av. Absorb. (595 nm) Prot. Conc. (mg/mL) Prot. Conc. (corrected)(mg/mL)
D9 0.542 1.470
D9 (1:5) 0.219 0.368 1.84
D12 0.541 1.466
D12 (1:5) 0.204 0.317 1.58

Related entries

Run 2: Ht31 Dose/Effect Curve & S0 D9/D12

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