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 Creating RII overlay protocol
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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):
Method
Creating radioactive RII units
- 1. Mix on ice the following
| Substance
|
Concentration
|
Volume
|
End amount
|
| RII(β) |
2.7 µg/µL |
5.6 µL |
15 µg+
|
| cPKA |
1.31 µg/µL |
2 µL |
2.6 µg+
|
| Potassiumphosphatebuffer (pH 7.0) |
1 M |
12.5 µL |
25 mM
|
| cAMP |
1 mM |
5 µL |
10 µM
|
| MgCl2 |
0.5 M |
10 µL |
10 mM
|
| Dithiothreitol |
50 mM |
5 µL |
0.5 mM
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[γ32P]-ATP = 3.3×108 cpm/mL
Specific activity 18.5 MBq
|
5 µCi/µL |
15 µL |
75 µCi
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| ATP (cold*) |
10 µM |
5 µL |
100 pM
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| [γ32P]-ATP/ATP (cold*) |
|
|
0.1 µM
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| H2O |
|
434.9 µL |
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* 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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Creating RII overlay protocol
