Differences in assay conditions (e.g., buffer pH, temperature) significantly impact results. For instance:
| Study | KD (nM) | Assay Conditions |
|---|---|---|
| A (2023) | 2.1 ± 0.3 | pH 7.4, 25°C |
| B (2024) | 5.8 ± 1.2 | pH 6.8, 37°C |
Standardize protocols using reference materials to minimize variability.
Molecular dynamics simulations (e.g., GROMACS) paired with cryo-EM data reveal allosteric binding sites. Key parameters include:
Force field: CHARMM36
Simulation time: ≥100 ns for convergence
Combine protease-induced epitope retrieval (PIER) with antibody dilution series. A ≥3-fold signal increase post-PIER suggests masking.
Use Arrhenius equation-based studies at 4°C, 25°C, and 40°C. Data trends indicate:
Activity loss follows first-order kinetics (R² = 0.96)
Activation energy (Ea): 85 kJ/mol