Western blot was conducted as described previously.19 (link) Briefly, cells were lysed and quantified using Bicinchoninic Acid Protein Assay Kit (Beyotime Biotechnology, Shanghai, P.R. China). Protein lysates (15 µg) were separated by 10% SDS-PAGE and transferred to Millipore PVDF membranes. Blots were blocked with 5% non-fat dry milk in Tris-buffered saline buffer for 2 hours at room temperature and then incubated with diluted antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Abcam, Cambridge, MA, USA) for 1 hour at room temperature. The blotting signal was visualized using an enhanced chemiluminescence detection reagent (Abcam). β-Actin served as a loading control.
Protocol detail
Western Blot Analysis of Protein Lysates
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Bicinchoninic Acid Protein Assay Kit
horseradish peroxidase-conjugated secondary antibody
enhanced chemiluminescence detection reagent
Actin
Antibodies
Antibody
Assay
Bicinchoninic acid
Cells
Chemiluminescence
Horseradish peroxidase
Membranes
Milk
Protein
Pvdf
Saline
Sds page
Western blot
Corresponding Organization :
Other organizations : Central South University, Xiangya Hospital Central South University
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Protocol cited in 2 other protocols
Variable analysis
independent variables
- Not explicitly mentioned
- Protein expression levels
- Cell lysis and protein quantification method (Bicinchoninic Acid Protein Assay Kit)
- Protein separation method (10% SDS-PAGE)
- Protein transfer method (Millipore PVDF membranes)
- Blocking buffer (5% non-fat dry milk in Tris-buffered saline buffer)
- Antibody incubation (diluted antibodies incubated overnight at 4°C)
- Secondary antibody incubation (horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature)
- Visualization method (enhanced chemiluminescence detection reagent)
- β-Actin served as a loading control
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