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RESEARCH DIVISION Laboratory Manual

 


 

Anti-Phosphotyrosine Western

  1. After SDS-PAGE, set up transfer as usual.
  2. Block for 1hr at RT before probing:

    Blocking solution:

      For 100 ml
    20mM Tris pH 7.4 2 ml 1M Tris pH 7.4
    150mM NaCl 3 ml 5M NaCl
    10% milk 10 g skim milk powder
    1% BSA 1 g BSA
    0.1% Tween 20 100 Ál Tween-20
    1mM vanadate 1 ml 100mM vanadate

    ddH20 to 100 ml

  3. The remaining steps are performed at RT in 5% milk, 20mM Tris pH 7.4, 150mM NaCl, 1mM vanadate, 0.1% Tween (no BSA).
    Stock solution For 500ml For 1 L
    1M Tris pH 7.4 10 ml 20 ml
    5M NaCl 15 ml 30 ml
    skim milk powder 25 g 50 g
    Tween 20 0.5 ml 1 ml
    sodium orthovanadate 0.09 g 0.18 g
    ddH20 to 500 ml 1 L
  4. Add antiphosphotyrosine antibody (UBI: 4G10) At 1/5000 (2ÁL/10ml) For 2 hr.
  5. Wash 6x 15 min.
  6. Incubate with anti-mouse-HRP conjugated secondary antibody for 1 hr.
  7. Wash 5x 10 min with 20mm Tris pH 7.4, 150mm NaCl, 0.1% Tween no milk).
  8. Blot membrane On 3mm paper.
  9. Place membrane in ECL mix for 1 min. Blot dry on 3mm paper.
  10. expose to film for 5 min, develop. Re-expose for various times as necessary.

 

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998