User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/24

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Co-IP Part III, Stimulation stop/Viability staining
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

 * Co-IP day III
 * Stop stimulation --> 12.41
 * Coat ELISA plate

Materials

 * Coating buffer
 * Solution A: 1.24 g Na2C03·H2O in 100 mL H2O
 * Solution B: 2.52 g NaHCO3 in 300 mL H2O
 * Take 70 mL of solution A
 * Add solution B until a pH of 9.6 is reached

Co-immunoprecipitation day III

 * Centrifuge sample incubated with beads, 5 min. 14.000 RPM
 * Remove supernatant* (w. syringe)
 * Add 1 mL PBS
 * Centrifuge, 5 min. 14.000 RPM
 * Remove supernatant*
 * Add 75 μL Laemli buffer to beads
 * Store samples @ -20 °C
 * Alternatively go directly on to Western blot (Boil samples and spin beads down, bring supernatant onto gel)


 * Be careful not to touch beads

Stop stimulation/Viability test

 * Collect 200 μL of supernatant for ELISA of each well
 * Remove the rest of the media
 * Wash twice with PBS
 * Add per well 475 μL HBSS + 25 μL Alamar blue
 * For ~50 wells that's 23.750 mL HBSS + 1250 μL Alamar blue
 * Incubate ~30 min. 37 °C
 * Measure in Wallace 1420 Victor 2TM, excitation 544 nm emission 390 nm
 * This shows the metabolic (mitochondria) activity of cells

Coating of plate

 * Mix twice
 * 24 mL coating buffer
 * 80 μL primary antibody
 * Add 100 μL per well (96-wells plate)
 * Incubate ON

Results

 * Viability staining

Related entries

 * Co-IP Day I & Cell S0
 * Co-IP Day II & Cell stimulation


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