User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/22

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Co-IP & Western blot
 * 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 I
 * Western blot Co-IP
 * Put cells to S0

Co-immunoprecipitation part I

 * Beads were coated with anti-PKA antibodies.
 * For 2 samples (D9/D12; Only Beads, Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample)
 * 280 μL beads
 * Spin down and remove EtOH
 * Wash with excess (400 μL) PBS
 * Spin down and remove PBS
 * Add 1:1 PBS:Beads (280 μL)
 * 200 μL for Beads with Antibody, Beads w. AB & RIPA and Bead w. AB & Sample (D9/D12)
 * 80 μL for Only Beads (D9/D12)
 * (30 μL for each condition)
 * Add 1:100 (PKA RIIβ) antibody (or not for control), 2 μL
 * Incubate ON @ 4 °C

SDS-PAGE

 * Clean the glass plates (UP, Soap, UP, EtOH and dry)
 * Prepare elaboration, look out for leakage!
 * Choose % of gel, big proteins require higher % (here 10% SDS gel)
 * Prepare separating gel (pour until green beam, ~10 mL per gel)
 * Add UP until flooded
 * When polymerized (approx. 30 min) remove isolating layer (remove UP with tissue paper)
 * Prepare 5% stacking gel
 * Pour until flooding and add combs
 * Dilute samples with the buffer which they are stored in until an equal amount is reached (20 ng in 30 μL)
 * Add 10 μL 4x loading buffer
 * Boil samples for 5 min. and cool samples on ice
 * Bring gels into the electrophoresis elaboration and pour ELFO buffer in the inner space to check for leakage
 * Remove combs and rinse slots with buffer
 * Fill slots with sample (40 μL) and marker (15 μL)
 * Start electrophoresis (40 min. 200 V)
 * Alternatively start with 100 V until samples reach the separating gel and increase voltage

Western Blot

 * Moisten sponges, filter papers and membrane in transfer buffer
 * Prepare sandwich
 * Black clamp
 * Sponge
 * Whatman paper
 * Gel
 * Membrane
 * Whatman paper
 * Sponge
 * White clamp
 * Put sandwhich in the transferring system (black to black) and fill it with transferbuffer
 * Transfer proteins to membrane for 1 hour @ 100 V on ice while stirring
 * After transfer check if marker is visible on membrane (successful transfer)
 * Alternatively Ponceau S staining is possible (not used @ this lab)
 * Remove LEFT UPPER corner to mark what is what
 * Block membrane with 5% milk solution in TBST buffer (ON, 4°C)
 * Alternatively 2 h @ RT
 * Treat with primary antibody (diluted in 5% TBST-milk solution) (ON, 4°C)
 * GAPDH 1:2000
 * VASP 1:1000
 * AKAP 1:1000
 * Wash 3x 10 min. in TBST buffer (first can be less)
 * Treat with secondary antibody (diluted in 5% TBST-milk solution) (1h, RT)
 * Wash 3x 10 min. in TBST buffer
 * 1) Only beads (30 μL)
 * 2) Beads w. antibody (30 μL)
 * 3) Beads w. antibody (30 μL) & lysis buffer (200 μL)
 * 4) Beads w. antibody (30 μL) & sample (200 μL)

A. 23Feb2010 to 25Feb2010 B. 03March2010 to 05March2010 C. 15March2010 to 17March2010

Related entries
A. 23Feb2010 to 25Feb2010 B. 03March2010 to 05March2010 C. 15March2010 to 17March2010

Same actions

 * Western blot
 * Co-IP day I


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