User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/04/01

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Creating RII overlay protocol Main project page
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Summary

RII subunits are autophosphorylated by the catalytic subunits with 32P. The radioactive RII subunits are used to hybridize with a western blot membrane.

Materials & Methods

Materials

1M Potassiumphosphatebuffer (pH 7.0)

  • 174.18 g K2HPO4
  • 136.09 g KH2PO4
  • Suspend in 800 mL of H2O
  • Set pH to 7.0
  • Add to 1 L H2O

Blocking solution (Pepperle):

  • 10 mM Potassiumphosphate buffer (pH 7.4)
  • 150 mM Sodiumchloride
  • 5% milkpowder
  • 0.1% BSA
  • 0.02% NaN3 (Sodiumazide)

Blocking solution (Stefan):

  • 5% milkpowder in PBS
  • 0.1% FBS
  • 0.02% Azide

Wash buffer (Pepperle):

  • 10 mM Potassiumphosphate buffer (pH 7.4)
  • 150 mM Sodiumchloride

Wash buffer (Stefan):

  • PBS


Method

Creating radioactive RII units

1. Mix on ice the following


Substance Concentration Volume End amount
RII(β)2.7 µg/µL5.6 µL15 µg+
cPKA1.31 µg/µL2 µL2.6 µg+
Potassiumphosphatebuffer (pH 7.0)1 M12.5 µL25 mM
cAMP1 mM5 µL10 µM
MgCl20.5 M10 µL10 mM
Dithiothreitol50 mM5 µL0.5 mM
32P]-ATP = 3.3×108 cpm/mL
Specific activity 18.5 MBq
5 µCi/µL15 µL75 µCi
ATP (cold*)10 µM5 µL100 pM
32P]-ATP/ATP (cold*)0.1 µM
H2O434.9 µL

* means unlabeled
+ end conc in 500 ml

2. Incubate on ice for 10 min.
3. Add 5 µL 1 mM ATP solution (ATP conc. now 10 µM)
4. Incubate for 50 min. on ice
5. Add 70 µL Dextran blue (of 20 mg/mL solution)
6. Remove excess nucleotides by running over a Sephadex G50-collumn

Removing excess nucleotides

7. 20 g Sephadex G50 material is incubated in 400 mL PBS ON @ RT
a. Remove material not settled using Pasteur pipette
b. Degassed material is stored @ 4 °C add for preservation 0.01% NaN3
8. The material is poured in a 10 mL sterile pipette (closed with glass marble)
9. Put 30-50 mL PBS with 1 mg/mL BSA through the column
10. Take 5.7 µL (1%) of sample for Build-in-rate
11. Put the rest of the sample over the collumn
12. Afterwards fill the column with PBS
13. Both Blue and clear fractions are collected
a. After eluting the blue front the column is washed with 2-3 mL PBS
b. Depending on the properties of the column, the RII subunits should run with the Dextran blue
14. To determine build-in-rate the 5.7 µL collected @ #10 and 1% (3 µL) of both fractions are used for liquid scintillation counting (2 mL scintillation fluid)


Hybridization

15. Incubate membrane ON @ 4 °C or 1h @ RT in blocking solution
16. Incubate membrane for 4-6 hours @ RT while shaking with 500 µL [32P]-RII subunits (10×106 cpm / membrane) (diluted in blocking solution)
17. Wash the membrane 4x 15 min. in blocking solution
18. Wash the membrane 2x 10 min. in wash buffer
19. Wrap the membrane in foil and prepare X-Rax (röntgen) film
a. Expose for 3 hours
b. Expose ON


References

I. Barbara Pepperle; Die Klonierung und Charakterisierung des protein kinase A anchoring protein r(rat)Ht31 aus der Rattennierre, Dissertation Freien Universität Berlin, 2002
II. Eduard Stefan, Beteiligung von cAMP-abhängigen Signaltransduktionskomponenter bei der Vasopressin-regulierten Umverteilung von Aquaporin-2 in renalen Hauptzellen, Dissertation Freien Universität Berlin, 2005
III. Christopher Blum; Funktionelle Analyse des Proteinkinase A Ankerproteins Ht31, Dissertation Freien Universität Berlin, 2005


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