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

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

Article DOI: 10.3791/1746-v 19:16 min March 17th, 2010
March 17th, 2010

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Summary

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.

Tags

Protein Concentration Kinetics Affinity Determination Structure-function Analysis Cysteine Protease Inhibitor Cystatin B Papain Real-time Label-free Analysis Biacore X100 Point Mutations Interaction Calibration-free Concentration Analysis (CFCA) A280 Single-cycle Kinetics (SCK) Association Rate Constant (ka) Affinity (KD) Reliable Results Interaction Mechanism
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