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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Creating RII overlay protocol
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |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
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

* 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