Methods have been described previously.15 (link)–17 (link) In brief, after each treatment, whole-cell lysates were prepared by sonication in Cellytic MT buffer (Sigma-Aldrich) with protease/phosphatase inhibitors (Cell Signaling Technology) and cleared by centrifugation. Samples consisting of 40 µg proteins were resolved on a denaturing 4%–20% SDS-PAGE gel (Bio-Rad) and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were then blocked in PBS blocking buffer (Li-Cor) for 1 hour and incubated with specific primary antibodies at 4°C overnight. Blots were then incubated with species-specific secondary antibodies (Cell Signaling Technology). Signals were detected by a chemiluminescence reagent and analyzed with ImageJ software.
Protocol detail
Protein Expression Analysis by Western Blot
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Cellytic MT buffer
protease/phosphatase inhibitors
4%–20% SDS-PAGE gel
PBS blocking buffer
species-specific secondary antibodies
Antibodies
Buffer
Cell
Centrifugation
Chemiluminescence
Membranes
Phosphatase
Polyvinylidene fluoride
Protease inhibitors
Proteins
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Corresponding Organization :
Other organizations : Fujian University of Traditional Chinese Medicine, First Hospital of Jilin University, Jilin University, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, First Affiliated Hospital of Fujian Medical University, Fujian Medical University
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Protocol cited in 2 other protocols
Variable analysis
independent variables
- Treatment
- Protein expression levels
- Protein amount (40 µg) per sample
- SDS-PAGE gel (4%-20%)
- Electroblotting to transfer proteins to PVDF membranes
- Blocking in PBS buffer for 1 hour
- Incubation with primary antibodies at 4°C overnight
- Incubation with secondary antibodies
- Detection using chemiluminescence reagent
- Positive control: Not specified
- Negative control: Not specified
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