For the detection of Bax and Bcl-2 expression, we used Western blotting, as the method described before [20 (link),21 (link)]. After separating 40-µg protein on sodium dodecyl sulfate-polyacrylamide gels, protein was transferred onto a nitrocellulose membrane (Schleicher & Schuell GmbH, Dassel, Germany). For the primary antibodies, we used mouse anti-actin antibody (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Bax antibody (1:1,000; Santa Cruz Biotechnology), and mouse antiBcl-2 antibody (1:1,000 Santa Cruz Biotechnology). As the secondary antibodies, we used mouse horseradish peroxidase-conjugated antibody (1:2,000; Santa Cruz Biotechnology) for actin, Bax, and Bcl-2. We used enhanced chemiluminescence detection system (Amersham Pharmacia Biotechnology GmbH, Freiburg, Germany) for the band detection. By Image-Pro Plus software (Media Cybernetics Inc.), the detected bands were densitometrically analyzed.
Protocol detail
Western Blot Analysis of Bax and Bcl-2
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nitrocellulose membrane
mouse anti-actin antibody
mouse anti-Bax antibody
mouse antiBcl-2 antibody
Image-Pro Plus software
Actin
Anti antibody
Antibodies
Antibody
Bcl 2
Chemiluminescence
Horseradish peroxidase
Membrane
Mouse
Nitrocellulose
Polyacrylamide gels
Protein
Sodium dodecyl sulfate
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Variable analysis
independent variables
- None explicitly mentioned
- Bax expression
- Bcl-2 expression
- Actin expression (used as a loading control)
- Positive control: None mentioned
- Negative control: None mentioned
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