Western blot procedures were as previously described (31 (link)). Briefly, membranes were blocked in PBST containing 3% milk for 1 hour at 20°C. Primary antibodies in PBST containing 3% milk were incubated at 4°C rocking overnight. The following morning membranes were washed 3x with PBST followed by the addition of secondary antibody in PBST containing 3% milk at 20°C for 2 hours. Membranes were then washed 3x with PBS. All western blot figures are representative data of at least 2 independent replicates. Band intensities were calculated using ImageJ (32 (link)). Antibodies used are: anti-p53 (1C12, Cell Signaling, Danvers, MA), anti-actin (Sigma), anti-GST (B-14, Santa Cruz, Dallas, TX), anti-His (Omni-probe D-8, Santa Cruz), anti-eIF4E (P-2, Santa Cruz), anti-p21 (H164, Santa Cruz), anti-p130 (A-10, Santa Cruz), anti-vinculin (7F9, Santa Cruz), anti-MDM2 (SMP14, Santa Cruz), and anti-HA (Covance, San Diego, CA).
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Comprehensive Western Blot Protocol
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Corresponding Organization : University of California, Davis
Other organizations : National Cancer Institute, The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Primary antibodies used (anti-p53, anti-actin, anti-GST, anti-His, anti-eIF4E, anti-p21, anti-p130, anti-vinculin, anti-MDM2, anti-HA)
- Band intensities of the proteins detected by the primary antibodies
- Membrane blocking conditions (PBST containing 3% milk for 1 hour at 20°C)
- Primary antibody incubation conditions (in PBST containing 3% milk at 4°C overnight)
- Secondary antibody incubation conditions (in PBST containing 3% milk at 20°C for 2 hours)
- Membrane washing conditions (3x with PBST, 3x with PBS)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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