User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/23

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Co-IP Part II, hTERT Stimulation & Western blot cont'd Main project page
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Summary

  • Cell lysis Co-IP
  • Co-IP Day II
  • Stimulation
    • ELISA
    • VASP
  • Western blot, secondary antibody

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)
  5. Beads (30 μL) with only sample (200 μL))
  • Incubate ON @ 4 °C

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 S0 (see schedule below)
    • Incubate 20 min.
  • 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
S0
1
S0
2
S0
3
HT31
4
HT31
5
HT31
6
A
(CTR)
B
(CSE)
C
(CSE+8-pcpt)
D
(CSE+6-Benz)

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 (anti-Mouse) (diluted in 5% TBST-milk solution) (1.5h, RT)
  • Wash 3x 10 min. in TBST buffer

Preparing film

  • 1:1 ratio ECL solutions (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
  • For Pierce determination new standard was made, only concentration 750, 500 & 250 μg/mL

Results

  • Co-IP Blots of samples pulled down using PKA antibody coated beads, blots were treated with anti-AKAP antibody


Gel 1

Gel 2

  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)

A. 23Feb2010 to 25Feb2010
B. 03March2010 to 05March2010
C. 15March2010 to 17March2010

Conclusion

  • All Co-IP samples incubated with sample (and antibody) showed a double band it is unclear wether or not it is at the right height. The used antibody was directed against AKAP179, 86 kDa

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