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

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Summary

  • Short summary

Materials & Methods

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

Method

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)
  • 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

  • WHAT?

--> PICTURES ANOUK

Conclusion

  • DONE Ow..

Related

Related topics

Western blot


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