User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/02/25

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Co-immunoprecipitation part III
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary
The samples have been incubated with the (PKA) antibody coated beads and today the beads are washed and stored in Laemli buffer

Materials

 * Laemli buffer
 * PBS
 * syringe
 * Hot lysis buffer

Co-immunoprecipitation

 * 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

VASP

 * Use cells grown by Bachelor students
 * Remove medium
 * Wash with PBS twice and remove
 * Add 600 μL S0 or S0+Ht31 (see hTERT VASP schedule below)
 * Incubate 15-20 min. RT
 * Add 600 μL S0 or S0+6-Bnz (see hTERT VASP schedule below)
 * Incubate 10 min. RT
 * Add 600 μL S0 or S0 45% CSE (see hTERT VASP schedule below)
 * Incubate 10 min. RT
 * Remove medium
 * Wash with PBS and remove
 * Add 200 μL Hot Lysis buffer and scrape cells with tip
 * Transfer cell lysate into 1.5 mL tubes
 * Store @ -20 °C

Coating of plate

 * Prepare coating buffer
 * 1.24 g Na2CO3 in 100 mL (Buffer A)
 * 2.52 g NaHCO3 in 300 mL (Buffer B)
 * Take 70 mL of Buffer A
 * Add Buffer B until pH of 9.6 has reached (150 - 200 mL)
 * Continue as described 18Feb2010

BSc.

 * Alamar blue
 * Stop stimulation (hTERT)
 * Cells to 96-wells (hTERT)

Results

 * Viability staining
 * Viability staining

Run 2: Ht31 Dose/Effect Curve & S0 D9/D12

 * Counting cells 19Feb2010
 * Putting to S0 23Feb2010
 * Stimulating cells 24Feb2010

Same action

 * ELISA

Related topics

 * SDS_sample_buffer
 * Sauer:bis-Tris_SDS-PAGE,_the_very_best
 * Odom:Western_Blot
 * Carrico:Recipes
 * Griffitts:Common_buffers
 * Griffin:Antibody_Related_Solutions_&_Recipes


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