The Western blotting was performed as previously described (Zhu et al. 2016 (link)). The expression of Flag-tagged or Myc-tagged exogenous proteins was detected by anti-Flag (Thermo Fisher Scientific Inc) and anti-Myc (Santa Cruz Biotechnology) antibodies respectively. Antibody for endogenous PRDX6 detection was purchased from Cell Signaling Technology. FMDV-positive serum (prepared by our lab previously) was used as the primary antibody to detect FMDV proteins. Rabbit anti-SVA-VP2 and SVA-3Cpro antibodies were prepared by our lab. The anti-rabbit, anti-mouse or anti-pig secondary antibodies conjugated with horseradish peroxidase were used for detection of the antigen–antibody complexes. Specific bands were visualized with enhanced chemiluminescent substrate (ECL). Each blot was stripped and reblotted with anti-β-actin (Sigma) antibody for loading control. Each immunoblot assay was carried out at least three times and one of them was presented.
Protocol detail
Western Blot Detection of Exogenous and Endogenous Proteins
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Actin
Anti antibodies
Antibodies
Antibody
Antigen antibody complexes
Fmdv
Horseradish peroxidase
Immunoblot assay
Mouse
Proteins
Rabbit
Serum
Corresponding Organization :
Other organizations : Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences
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Variable analysis
independent variables
- Expression of Flag-tagged or Myc-tagged exogenous proteins
- Detection of Flag-tagged or Myc-tagged exogenous proteins
- Detection of endogenous PRDX6
- Detection of FMDV proteins
- Detection of SVA-VP2 and SVA-3C^pro
- Anti-Flag and anti-Myc antibodies
- Anti-PRDX6 antibody
- FMDV-positive serum
- Rabbit anti-SVA-VP2 and SVA-3C^pro antibodies
- Anti-rabbit, anti-mouse, or anti-pig secondary antibodies conjugated with horseradish peroxidase
- Anti-β-actin antibody for loading control
- FMDV-positive serum for detection of FMDV proteins
- Not explicitly mentioned
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