Follow general Western Blot Protocol up to gel transfer step and proceed as follows:
- 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.
- Wash the membrane three (3) times for 5 minutes each in TBST.
- 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.
- Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
- Wash the membrane three (3) times for 10 minutes each in TBST.
- 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.
- Incubate the membrane in diluted streptavidin-HRP at room temperature for 60 minutes.
- Wash as directed in step 5.
- Develop blots with substrate solution (e.g. Super Signal West Dura, Thermo Scientific Cat# 34075) and place in plastic membrane protector.
- Expose membrane to film or CCD camera.
