User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/15: Difference between revisions
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** 1.06 mg β-glycerolphosphate | ** 1.06 mg β-glycerolphosphate | ||
** 1 mL [http://wikilaboratory.com/index.php?title=RIPA_Buffer RIPA buffer] | ** 1 mL [http://wikilaboratory.com/index.php?title=RIPA_Buffer RIPA buffer] | ||
** 1 μL Apoprotein | ** 1 μL Apoprotein (1 mg/mL) | ||
** 1 μL Leupeptin | ** 1 μL Leupeptin (1 mg/mL) | ||
** 1 μL Pepstatin (A) | ** 1 μL Pepstatin (A) (1 mg/mL) | ||
** 5 μL Na<sub>3</sub>VO<sub>4</sub> | ** 5 μL Na<sub>3</sub>VO<sub>4</sub> | ||
** 5 μL NaF | ** 5 μL NaF (200 mM) | ||
* [[PBS]] | * [[PBS]] | ||
* DMEM S<sup>0</sup> | |||
*[http://www.piercenet.com/Products/Browse.cfm?fldID=02020105 Coomassie (Bradford) Protein Assay Kit] | *[http://www.piercenet.com/Products/Browse.cfm?fldID=02020105 Coomassie (Bradford) Protein Assay Kit] | ||
** [http://www.piercenet.com/Products/Browse.cfm?fldID=02020101 BCA Protein Assay Reagent (bicinchoninic acid)] | ** [http://www.piercenet.com/Products/Browse.cfm?fldID=02020101 BCA Protein Assay Reagent (bicinchoninic acid)] | ||
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===Method=== | ===Method=== | ||
====Putting cells to S<sup>0</sup>==== | |||
* Wash cells twice with PBS | |||
* Add 500 μL DMEM S<sup>0</sup> to each well | |||
* Incubate @ 37 °C | |||
====[http://genome.med.yale.edu/images/a/aa/BCA.pdf Determination of protein concentration]==== | ====[http://genome.med.yale.edu/images/a/aa/BCA.pdf Determination of protein concentration]==== | ||
* Use cells on S<sup>+</sup> refreshed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| 12March2010]] | * Use cells on S<sup>+</sup> refreshed [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/12| 12March2010]] | ||
** 1 Ø 10 cm dish each donor | |||
* Put cells on ice | * Put cells on ice | ||
* Remove medium | * Remove medium | ||
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* Sonicate (4x short pulse) | * Sonicate (4x short pulse) | ||
* Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination) | * Take 10 μL of undiluted and a 5x diluted samples for Bradford assay (Pierce determination) | ||
* Store samples @ -20 °C | |||
====Co-immunoprecipitation part I==== | ====Co-immunoprecipitation part I==== | ||
* Beads were coated with anti-PKA antibodies. | * Beads were coated with anti-PKA antibodies. | ||
* For <b> | * For <b>8</b> samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample) | ||
** | ** 960 μL beads | ||
** Spin down and remove EtOH | ** Spin down and remove EtOH | ||
** Wash with excess ( | ** Wash with excess (800 μL) PBS | ||
** Spin down and remove PBS | ** Spin down and remove PBS | ||
** Add | ** Add 800 μL PBS | ||
** Add | ** Add 0.6 μL antibody/120 μL (or not for control) | ||
** Incubate with antibody ON @ 4 °C | ** Incubate with antibody ON @ 4 °C | ||
===Coating of ELISA plate=== | ===Coating of ELISA plate=== | ||
* Mix | * Mix | ||
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==Results== | ==Results== | ||
*{{todo| | *{{todo| PIERCE}} | ||
==Conclusion== | ==Conclusion== | ||
Revision as of 05:36, 15 March 2010
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Summary
Materials & MethodsMaterials
MethodPutting cells to S0
Determination of protein concentration
Co-immunoprecipitation part I
Coating of ELISA plate
NotesResults
Conclusion
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