Biochemistry JoVE (Open Access) Citable · DOI

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

Name: The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Uploaded: 2026-06-17
Description: In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

DOI: 10.3791/1746-v

What you'll learn

✓ Apply calibration-free concentration analysis (CFCA) to measure active protein concentration
✓ Determine kinetic constants reliably using surface plasmon resonance (Biacore X100)
✓ Validate protein-ligand binding kinetics despite reduced recombinant protein activity

Protocol

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

Difficulty

advanced

Total time

~4–6 hours per sample set (including ligand immobilization, concentration determination, and kinetic measurements)

Biosafety

BSL-1

Steps

Review experimental design and CFCA methodology

Understand the rationale for using calibration-free concentration analysis to confirm active protein concentration and improve kinetic reliability. Review the workflow for structure-function analysis of cystatin B mutants.

▶ 02:30

Immobilize papain ligand on sensor chip

Prepare and immobilize the papain ligand onto the Biacore X100 sensor chip surface using standard amine-coupling chemistry to establish the interaction surface.

▶ 03:58

Determine cystatin B concentration using CFCA

Apply the calibration-free concentration analysis assay to measure the concentration of cystatin B variants with retained binding activity, without requiring a standard curve.

▶ 07:34

Measure kinetic binding of cystatin B to papain

Perform surface plasmon resonance kinetic analysis using the confirmed protein concentration to determine accurate association and dissociation rate constants.

▶ 11:07

Analyze CFCA and kinetic binding results

Evaluate representative kinetic data and CFCA outputs to demonstrate reliability of concentration confirmation and accuracy of determined kinetic parameters.

▶ 17:13

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