Octet® Label-Free Applications Guide
Designing and performing assays that accurately measure the binding kinetics of biomolecular interactions can seem too specialized and out of reach for many scientists.
This applications guide aims to change that by serving as an easy-to-use reference for basic assay design on both BLI and SPR systems. The chapters are designed to answer specific questions about these techniques, complete with relevant application notes and best practices so users are empowered to get the best data in the shortest time possible.
We understand that label-free biomolecular interaction analysis requires more than a product. It requires a strategy.
Page Count: 104Read Time: 2 hoursLast Updated: March 2023
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1. Why Are Real-Time Kinetics and Affinity Important?
1.0.1 The Advantages of Real-Time and Label-Free Information
1.1 What Is in the Octet® Range?
1.1.1 Why Choose Octet®?
1.1.2 GxP-Ready Systems
2. What Can Label-Free, Real-Time Assays Tell You?
2.0.1 Binding Kinetics
2.0.2 Steady State Affinity
2.0.3 Sample Quantification
2.0.4 Epitope Sites
2.0.5 Dose-Response Curves
2.1 Where Can You Use Label-Free, Real-Time Assays?
2.1.1 Stage: Target ID and Validation—ELISA Assay Development
2.1.2 Stage: Lead Screening and Selection—Epitope Binning
2.1.3 Stage: Process Development—Monitoring Titer and Glycosylation
3.1 Principles of Label-Free Analysis
3.1.1 Baseline
3.1.2 Loading
3.1.3 Association
3.1.4 Dissociation
3.1.5 Regeneration
3.1.6 Putting It All Together
3.2 How Does BLI Work?
3.3 How Does SPR Work?
3.4 Overview of Kinetics and Affinity
3.4.1 Multi-Cycle Kinetics
3.4.2 Steady State Affinity
3.4.3 OneStep® Injections
4.1 What to Consider Before Starting Experimental Design
4.2 Assigning the Ligand and Analyte
4.3 Biosensor and Sensor Chip Selection
4.3.1 How to Choose a Suitable Attachment Approach for Your Ligand
4.3.2 Covalent Immobilization
4.3.3 Affinity Capture Approaches
4.3.4 Streptavidin-Based Capture
4.4 Ligand Optimal Density Scouting
4.4.1 pH Scouting
4.4.2 How Much to Immobilize or Capture?
4.4.3 Kinetic Assays
4.4.4 Concentration Assays
4.5 Sensor Conditioning
4.5.1 The Importance of Sensor Hydration and Achieving a Stable Baseline
4.5.2 BLI Biosensor Hydration
4.5.3 SPR Sensor Chip Hydration
4.6 Assay Buffer Optimization
4.7 Analyte Association and Dissociation Optimization
4.7.1 Kinetic Screening and Dealing with Unknown KDs
4.7.2 Choosing the Optimal Concentration Series
4.7.3 Dealing With High-Affinity Binders
4.7.4 Optimizing the Dissociation Step
4.8 Surface Regeneration, Ligand Regeneration | Scouting
4.8.1 Determining Suitable Regeneration Conditions
4.8.2 Issues Affecting Regeneration
4.8.3 Determining Regeneration Conditions
4.8.4 Interpreting Experiments to Optimize Regeneration
4.9 Non-Specific Binding
4.9.1 Electrostatic and Non-Electrostatic Non-Specific Binding
4.9.2 Assay Design to Minimize Non-Specific Binding
4.10 General Assay Optimization
4.10.1 Reference Surface
4.10.2 Shake Speed
4.11 Mass Transport Limitation
4.12 Double Reference Subtraction
4.12.1 The Purpose of Referencing
4.12.2 Double Reference Subtraction
4.12.3 Solution Types
4.12.4 Refractive Index
4.12.5 Solvent Correction Curves
4.12.6 Preparing Running Buffer and Calibration Solutions
5.1 Initial Visual Data Evaluation
5.1.1 Buffer Blanks
5.1.2 Binding Data
5.1.3 Kinetics or Affinity?
5.1.4 Choosing an Appropriate Model
5.1.5 Visual Assessment of Data Fitting
5.2 Binding Models
5.2.1 1:1 Kinetics
5.2.2 1:1 Kinetics—OneStep®
5.2.3 Steady State/Equilibrium Analysis
5.2.4 Dissociation Kinetics
5.2.5 2:1 Heterogeneous Ligand
5.2.6 Mass Transport
5.2.7 1:2 Bivalent Analyte
5.2.8 Two-State (SPR Only)
5.2.9 Diffusion (SPR Only)
5.2.10 Aggregation (SPR Only)
6.1 BLI Information
6.1.1 Videos
6.1.2 Application Guides
6.1.3 Application Notes
6.1.4 Brochures
6.1.5 Case Studies
6.1.6 eBooks
6.1.7 Editorials
6.1.8 Flyers
6.1.9 Infographics & Posters
6.1.10 Technical Notes
6.1.11 Webinars
6.1.12 White Papers
6.2 SPR Information
6.2.1 Application Notes
6.2.2 Best Practice Guides
6.2.3 Brochures
6.2.4 Flyers
6.2.5 Technical Note
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