📋 Workflow
Full workflow, products add-to-cart directly1
Stimulation and sample collection
30 minTreat cells and stimulate over time course, quench and collect lysates
💡 Tip Include unstimulated and time-point controls
⚠️ Caution Quench quickly to preserve phosphorylation
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2
Protein quantification and normalization
1 hQuantify protein and load equal amounts for comparable signals
💡 Tip Equal loading for reliable quantification
⚠️ Caution Quantification errors bias pathway comparison
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3
Western blot for phosphorylation
1 daySeparate by SDS-PAGE, transfer, blot with phospho-specific and total protein antibodies
💡 Tip Compare matched phospho/total antibodies
⚠️ Caution Normalize to reference/total protein
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4
Inhibitor validation and integration
1 dayUse pathway inhibitors to confirm causality, integrate data and map the pathway
💡 Tip Cross-validate with multiple kinase inhibitors
⚠️ Caution Confirm inhibitor selectivity
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⚠️ 5 Common Beginner Mistakes
- ① Delayed quenching loses phosphorylation
- ② Poor antibody specificity gives false bands
- ③ Uneven loading biases comparison
- ④ Excess inhibitor causes off-target effects
- ⑤ Missing normalization internal/total control
❓ FAQ
+How to choose phospho-antibodies?
Choose validated antibodies specific to the phospho-site, with total protein controls.
+Why time course?
Signaling is dynamic; time course reveals activation kinetics and peaks.
+How do inhibitors validate pathway?
If downstream phosphorylation drops after upstream kinase inhibition, it confirms cascade dependency.