Briefly, liver tissue was snap frozen in liquid nitrogen. Tissue was homogenized in lysis buffer, and supernatants were quantified for protein amount (18 (link)). 160 ug of cell lysate was loaded in 10% SDS-PAGE, transferred to a nitrocellulose membrane and incubated with primary antibody overnight at 4° C (1000x dilution for anti-p53 (Novus), 2500x anti-GAPDH, 5000x anti-mouse IgG-HRP (Santa Cruz), 500x anti-pERK1/2 (Santa Cruz), 500x anti-MDM2 (Santa Cruz).
Protocol detail
Western Blot Analysis of Liver Tissue
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Anti igg
Antibody
Buffer
Cell
Dilution
Frozen
Gapdh
Liver
Mdm2
Membrane
Mouse
Nitrocellulose
Nitrogen
Novus
Protein
Sds page
Tissue
Corresponding Organization : The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Tissue homogenization in lysis buffer
- Protein amount
- Protein expression levels of p53, GAPDH, pERK1/2, and MDM2
- Snap freezing of liver tissue in liquid nitrogen
- Protein loading amount (160 ug of cell lysate)
- SDS-PAGE gel concentration (10%)
- Antibody dilutions (1000x for anti-p53, 2500x for anti-GAPDH, 5000x for anti-mouse IgG-HRP, 500x for anti-pERK1/2 and anti-MDM2)
- Overnight incubation with primary antibodies at 4°C
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