Validation Strategy Overview
ABPP-WB and CETSA-WB are two complementary target validation methods that independently verify ABPP or CETSA experiment results through Western Blot detection, ensuring reliability of target identification.
ABPP-WB Validation
- Label cell/tissue samples with reporter-group-conjugated ABPP probes
- After SDS-PAGE separation and transfer, detect via anti-reporter antibodies (e.g., streptavidin-HRP)
- Visually compare target protein labeling intensity differences between treatment groups
- Suitable for rapid validation of targets with known molecular weight
CETSA-WB Validation
- After gradient heating, centrifuge to separate soluble and precipitated fractions
- Western Blot detects soluble target protein levels at different temperatures
- Plot melting curves to calculate Tm shift (ΔTm)
- ΔTm > 2 °C indicates positive binding result
Combined Validation Workflow
We recommend a cascading validation strategy: "ABPP discovery → ABPP-WB preliminary validation → CETSA-WB cellular validation" to minimize false-positive risk.