After predicting peptides using docking software, what should be used to validate the binding?

After predicting the binding mode of a peptide with a target protein through molecular docking, it is necessary to experimentally verify its binding affinity and specificity. Common methods can be divided into two categories:

 

1. Biochemical or biophysical methods (direct measurement of binding)

1. Surface Plasmon Resonance (SPR)

Can obtain real-time binding kinetic parameters (ka, kd) and the equilibrium dissociation constant (KD), suitable for verifying affinity and kinetic characteristics.

 

2. Isothermal Titration Calorimetry (ITC)

Can directly measure thermodynamic parameters of binding (ΔG, ΔH, ΔS) and affinity, suitable for high-affinity systems.

 

3. Microscale Thermophoresis (MST)

Requires a low amount of sample and can quickly assess the binding constant.

 

4. Fluorescence Polarization (FP) or FRET

Can be used for screening small molecule/peptide and protein binding but requires labeling.

 

2. Cell or functional level validation (indirect evidence)

1. Co-immunoprecipitation (Co-IP) or Pull-down assays

Verify whether the peptide and protein form a complex in cells or in vitro.

 

2. Functional assays (activation/inhibition of signaling pathways, enzyme activity inhibition, etc.)

Verify whether the binding has a biological effect.

 

Recommendations

• In the initial screening phase, use MST or SPR to evaluate the actual affinity of the peptide with the target and compare it with the docking prediction results.

• Use ITC to accurately measure thermodynamic parameters for high-priority candidates.

• If the binding is stable and has biological function, further verify physiological relevance through cellular functional assays.

 

Biotech Pioneer - Characterization of Biologics, Premium Multi-omics Mass Spectrometry Analysis Service Provider

 

Related services:

CO-IP Immunoprecipitation Protein Interaction Analysis