Membrane and tissue homogenates were prepared as previously described.19 (link),50 (link) Briefly, Proteins were extracted by using total protein Extraction Kit (Millipore; Darmstadt, Germany). Protein concentration was determined using a Bradford assay. In all, 30 μg of proteins were then run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred on polyvinylidene difluoride membrane. Following primary antibodies were used for immunoblotting: GSK3β, GSK3β-Phosph(ser9), p53, Bax, CXCR4, SDF-1, and GAPDH. Proteins were visualized using HRP-conjugated secondary antibodies and exposed using chemiluminescent HRP substrate (Millipore, MA USA).
Protocol detail
Protein Expression Analysis in Tissue
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Antibodies
Assay
Cxcr4
Gapdh
Gels
Gsk3β
Membrane
Polyvinylidene difluoride
Proteins
Sdf 1
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Tissue
Corresponding Organization :
Other organizations : Icahn School of Medicine at Mount Sinai
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Variable analysis
independent variables
- Protein extraction method (using total protein Extraction Kit)
- Protein expression levels of GSK3β, GSK3β-Phosph(ser9), p53, Bax, CXCR4, SDF-1, and GAPDH
- Protein concentration (determined using a Bradford assay)
- Protein loading (30 μg of proteins per lane)
- Not explicitly mentioned
- Not explicitly mentioned
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