Protein extracts were prepared with RIPA buffer containing a mixture of protease inhibitors as described.23 (link) Briefly, 50 μg of protein was applied to a 12% SDS/PAGE and transferred to nitrocellulose membranes. Membranes were incubated with Odyssey blocking buffer (Li-Cor) prior to incubation with polyclonal or monoclonal antibodies to phospho-JNK, p53, caspase3, or β-actin overnight at 4°C. Goat anti-rabbit IgG (HþL) 800 CW and/or goat anti-mouse (HþL) was applied for 60 minutes at room temperature (1:25,000, LI-COR) prior to washing with 1X Phosphate Buffered Saline Tween-20 (PBS-T). Visualization and quantification were carried out with the LI-COR OdysseyH scanner and software (LI-COR Biosciences).
Protocol detail
Western Blot Analysis of Cellular Signaling
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Actin
Anti igg
Caspase3
Goat
Membranes
Monoclonal antibodies
Mouse
Nitrocellulose
Phosphate
Protease inhibitors
Protein
Rabbit
Ripa
Saline
Sds page
Tween 20
Corresponding Organization : Lehman College
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Variable analysis
independent variables
- None explicitly mentioned
- Phosphorylation of JNK
- P53 levels
- Caspase-3 levels
- Beta-actin levels
- Protein extract preparation with RIPA buffer containing protease inhibitors
- Protein amount (50 μg) applied to 12% SDS/PAGE
- Transfer to nitrocellulose membranes
- Blocking with Odyssey blocking buffer (Li-Cor)
- Incubation with polyclonal or monoclonal antibodies to phospho-JNK, p53, caspase3, or β-actin overnight at 4°C
- Incubation with goat anti-rabbit IgG (HþL) 800 CW and/or goat anti-mouse (HþL) for 60 minutes at room temperature (1:25,000, LI-COR)
- Washing with 1X Phosphate Buffered Saline Tween-20 (PBS-T)
- Visualization and quantification with the LI-COR OdysseyH scanner and software (LI-COR Biosciences)
- None explicitly mentioned
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