The entire IVC was removed with care taken to avoid surrounding arterial and connective tissues. Proteins were extracted with RIPA lysis buffer containing protease inhibitors from single IVC.11 (link), 13 (link), 21 (link) Protein concentrations were assessed using a colorimetric assay (Bio Rad), and equal amounts of protein were run on a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk, membranes were probed overnight with the primary antibodies (Major Resources Table ). After overnight incubation, the membranes were incubated with HRP conjugated secondary antibodies for 1 hour at room temperature and were developed with using Western Lightning Plus ECL reagent (NEL105001EA, PerkinElmer; Waltham, MA).
Protocol detail
Western Blotting of IVC Proteins
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Antibodies
Arterial
Assay
Buffer
Colorimetric
Connective tissues
Link protein
Membranes
Milk
Polyvinylidene difluoride
Protease inhibitors
Protein
Ripa
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Corresponding Organization :
Other organizations : Kyushu University, Capital Medical University, The University of Tokyo, Kyushu Central Hospital of the Mutual Aid Association of Public School Teachers, Nagoya University, VA Connecticut Healthcare System, Yale University
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Variable analysis
independent variables
- Removal of the entire IVC with care taken to avoid surrounding arterial and connective tissues
- Protein concentrations assessed using a colorimetric assay (Bio Rad)
- Equal amounts of protein run on a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and then transferred to a polyvinylidene difluoride membrane
- Protein expression levels detected by probing with primary antibodies and HRP conjugated secondary antibodies, and developed using Western Lightning Plus ECL reagent
- Protease inhibitors added to the RIPA lysis buffer used to extract proteins from the single IVC sample
- No positive or negative controls were explicitly mentioned in the input protocol.
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