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Protocols

Western Blot Protocol

Follow general Western Blot Protocol up to gel transfer step and proceed as follows:

  1. Remove the membrane from the transfer apparatus and place in 20 ml of 1% non-fat dry milk in TBST for one hour at room temperature, with gentle shaking.
  2. Wash the membrane three (3) times for 5 minutes each in TBST.
  3. Dilute the primary biotin-conjugated antibody in 15 ml of 1% non-fat dry milk in TBST. For best results, the optimal dilution of antibody should be empirically defined.
  4. Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
  5. Wash the membrane three (3) times for 10 minutes each in TBST.
  6. Dilute streptavidin-HRP conjugate (Thermo Scientific, Rockford, IL, Prod# 21130) in 15 ml of 1% non-fat dry milk in TBST. Typical dilutions are in the range of 1:5000 to 1:15,000 of a 1 mg/ml stock. For best results, the optimal concentration of streptavidin-HRP should be empirically defined.
  7. Incubate the membrane in diluted streptavidin-HRP at room temperature for 60 minutes.
  8. Wash as directed in step 5.
  9. Develop blots with substrate solution (e.g. Super Signal West Dura, Thermo Scientific Cat# 34075) and place in plastic membrane protector.
  10. Expose membrane to film or CCD camera.