Cellular protein in the transfected cells was extracted by RIPA lysis buffer (Beyotime, Shanghai, P.R. China). Protein samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk for 1 h at room temperature and were incubated with the special primary antibodies: caspase 8 (1C12), cleaved caspase 8 p18 (Asp387), or GAPDH (D4C6R) (all at a dilution of 1:1,000; Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein bands were developed using enhanced chemiluminescence and analyzed by ImageJ 1.49 (National Institutes of Health, Bethesda, MD, USA)19 (link).
Protocol detail
Western Blot Analysis of Caspase Activation
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Antibodies
Antibody
Buffer
Caspase 8
Cellular
Chemiluminescence
Dilution
Gapdh
Horseradish peroxidase
Membranes
Milk
Polyvinylidene fluoride
Protein
Ripa
Sds page
Corresponding Organization :
Other organizations : Third Hospital of Hebei Medical University, Hebei Medical University
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Variable analysis
independent variables
- Cellular protein in the transfected cells
- Protein bands developed using enhanced chemiluminescence and analyzed by ImageJ 1.49
- RIPA lysis buffer (Beyotime, Shanghai, P.R. China)
- 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
- Polyvinylidene fluoride membranes
- 5% skim milk for 1 h at room temperature
- Special primary antibodies: caspase 8 (1C12), cleaved caspase 8 p18 (Asp387), or GAPDH (D4C6R) (all at a dilution of 1:1,000; Cell Signaling Technology, Beverly, MA, USA)
- Horseradish peroxidase-conjugated secondary antibody
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