User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/01

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] 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

 * Short summary

Materials

 * Transfer buffer (1 L)
 * 700 mL H2O (Cold)
 * 100 mL 10x Transfer buffer (Cold)
 * 0.25 M TRIS (30.3 g/L)
 * 1.92 M Glycin (144.1 g/L)
 * 1.0% SDS (10 g/L)
 * 200 mL Methanol (Cold)
 * MIX and Store @ 4 °C
 * TBST buffer (4 L)
 * 3.6 L UltraPure H2O
 * 400 mL 10x TBS
 * for 1 L
 * 60.5 g/L TRIS
 * 87.6 g/L NaCl
 * pH 7.6
 * 4 mL Tween20
 * Mix
 * 1x ELFO buffer
 * 10x ELFO buffer
 * 30.26 g/L TRIS
 * 187.60 g/L Glycin
 * 10 g/L SDS

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 200 μL of isobutanol OR UP until flooded
 * Use Pierce determination to load equally
 * When polymerized (approx. 30 min) remove isolating layer (wash 3x with UP when isobutanol, 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
 * DON'T FORGET ICE BLOCK!
 * 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

Preparing film

 * 1:1 ratio ECL solutions (2 mL per membrane)
 * Incubate membrane in ECL solution 5 min, RT (both sides of membrane)
 * Place blot in between two overhead sheets
 * Take picture (depends on what is available)

Results
--> PICTURES ANOUK

Conclusion

 * ✅ Ow..

Related topics
Western blot


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