Methods for Fragments Screening Using Surface Plasmon Resonance

✍ Scribed by Sameer Mahmood Zaheer (editor), Ramachandraiah Gosu (editor)

• Publisher: Springer
• Year: 2021
• Tongue: English
• Leaves: 116
• Category: Library

✦ Synopsis

This volume describes methods and protocols for the fragment-based screening of proteins using Surface Plasmon Resonance (SPR). The initial chapter of the book discusses the principle of SPR for the identification of biomolecular interactions, while the subsequent chapters introduce methods for labelling proteins with different tags including, histidine and biotin tags. It also discusses techniques and factors that affect the amine and biotin-streptavidin coupling and methods to optimize the interactions. Next, it describes fragment preparation for screening in SPR and presents methods to calculate equilibrium dissociation constant (KD) and ligand efficiency (LE). It reviews techniques of next-generation injections that improve the efficiency of the characterization process over traditional SPR by determining the kinetics and affinity in a single step. Finally, the book elucidates a comprehensive yet representative description of challenges associated with the molecular interaction of proteins using SPR.

✦ Table of Contents

Preface
Acknowledgments
Contents
Editors and Contributors
Abbreviations
1: Introduction to Surface Plasmon Resonance
1.1 Introduction
References
2: Principle of Surface Plasmon Resonance (OneStep)
2.1 Principle
Part I: SPR Compatible Protein Preparation
3: Preparation of Protein with Histidine Tag for Amine Coupling
3.1 Introduction
3.2 Materials
3.2.1 Cloning and Expression
3.2.2 Purification of Proteins
3.2.3 Buffers Component Required for Protein Purification
3.2.4 2xYT Media Preparation
3.2.5 Buffer Stocks Required for Protein Purification
3.2.6 Resuspension/Binding Buffer (1000 mL)
3.2.7 Ni-Affinity Wash Buffer (1000 mL)
3.2.8 Ni-Affinity Elution Buffer (1000 mL)
3.2.9 Desalting/SEC Buffer (1000 mL)
3.2.10 Dialysis Buffer (1000 mL)
3.3 Methods
3.3.1 Gene Synthesis and Cloning
3.3.2 Transformation and Expression
3.3.3 Purification Process
3.3.3.1 Lysate Preparation
3.3.3.2 Column Packing
3.3.3.3 Akta Pure FPLC System Wash
3.3.3.4 Chromatography
3.3.3.5 Dialysis
3.4 Notes
References
4: Preparation of Protein with AviTag for Biotin-Based Capture
4.1 Introduction
4.2 Materials
4.2.1 Cloning and Expression
4.2.2 Purification of Proteins
4.2.3 Buffers Component Required for Protein Purification
4.2.4 2xYT Media Preparation
4.2.5 Buffer Stocks Required for Protein Expression and Purification
4.2.6 Preparation of Biotin (5 mM)
4.2.7 Resuspension/Binding Buffer (1000 mL)
4.2.8 Ni-Affinity Wash Buffer (1000 mL)
4.2.9 Ni-Affinity Elution Buffer (1000 mL)
4.2.10 Desalting/SEC Buffer (1000 mL)
4.2.11 Dialysis Buffer (1000 mL)
4.3 Methods
4.3.1 Cloning and Expression
4.3.2 Purification Process
4.3.2.1 Lysate Preparation
4.3.2.2 Column Packing
4.3.2.3 Akta Pure FPLC System Wash
4.3.2.4 Chromatography
4.3.2.5 Dialysis
4.4 Notes
References
Part II: Characterization of Protein for SPR
5: Protein Stability Using Thermal Shift Assay (TSA): pH Tolerance
5.1 Introduction
5.2 Materials
5.2.1 Components Required for TSA
5.2.2 Buffer and Reagents for TSA
5.2.3 Prepation of Sypro Orange Dye (50x)
5.3 Methods
5.3.1 Priming TSA
5.3.2 Procedure in Setting up TSA Experiment
5.3.3 Analysis
5.4 Notes
References
6: Protein Stability Using Thermal Shift Assay (TSA): DMSO Tolerance
6.1 Introduction
6.2 Materials
6.2.1 Reagents Required for TSA
6.2.2 Buffer and Reagent Preparation for TSA
6.2.3 Prepation of Protein Storage Buffer (50 mL)
6.2.4 Prepation of Protein or Ligand Stock (50 uM)
6.2.5 Prepation of Sypro Orange Dye (50x)
6.3 Methods
6.3.1 Priming TSA
6.3.2 Procedure for Setting Up TSA Experiment
6.3.3 Results and Analysis
6.4 Notes
References
Part III: Fragment Screening Using SPR
7: Selection and Identification of Fragment Library
7.1 Introduction
References
8: Preparation of SPR Sensor
8.1 Introduction
8.2 Materials
8.2.1 Immobilization Buffer
8.2.2 Preparation of 0.4 M EDC
8.2.3 Preparation of 0.1 M NHS
8.2.4 Preparation of Ligand for Immobilization
8.2.5 Preparation of Blocking Reagent
8.2.6 Preparation of of 20% DMSO
8.2.7 Preparation of of 10 mM DTT
8.2.8 Preparation of of 0.05% Tween20
8.2.9 Preparation of 1000 mL Running Buffer (1x PBS)
8.2.10 Maintenance Sensor
8.2.11 Cleaning Cartridge
8.3 Methods
8.3.1 Preinstallation Procedure
8.3.2 CDH Sensor
8.3.2.1 Activation of Sensor
8.3.2.2 Loading of Ligand onto the Sensor
8.3.2.3 Blocking of Ligand onto Sensor
8.3.2.4 Stabilization of Ligand onto Sensor
8.3.3 SADH Sensor
8.3.3.1 Equilibration of Sensor
8.3.3.2 Loading of Ligand onto the Sensor
8.3.3.3 Blocking of Ligand onto Sensor
8.3.3.4 Stabilization of Ligand onto Sensor
8.4 Notes
References
9: Optimization and Validation of Amine Coupling of Ligand
9.1 Introduction
9.2 Materials
9.2.1 30% DMSO Solution (100 mL)
9.2.2 20% DMSO Solution (100 mL)
9.2.3 10% DMSO Solution (100 mL)
9.2.4 10 mM DTT (100 mL)
9.2.5 0.05% Tween20 (100 mL)
9.2.6 3% DMSO in 10 mM HEPES Buffer (10 mL)
9.2.7 2% DMSO in 10 mM HEPES Buffer (1000 mL)
9.2.8 1% DMSO in 10 mM HEPES Buffer (10 mL)
9.2.9 0% DMSO in 10 mM HEPES Buffer (50 mL)
9.2.10 1.5% DMSO in 10 mM HEPES Buffer (10 mL)
9.2.11 2.8% DMSO in 10 mM HEPES Buffer (10 mL)
9.2.12 3% Sucrose in 10 mM HEPES Buffer (10 mL)
9.2.13 Concentrations of Small Molecules Required (Reference)
9.2.14 Preparation of 10 mM Master Stock
9.2.15 Preparation of 0.5 mM Substock
9.2.16 Preparation of 1.25 mM Substock
9.2.17 Preparation of 2.5 mM Substock
9.2.18 Preparation of 10 uM Working Concentration
9.2.19 Preparation of 25 uM Working Concentration
9.2.20 Preparation of 50 uM Working Concentration
9.2.21 SPR Compatible 96 Well Plate
9.3 Methods
9.3.1 Refractive Index (RI) Transfer
9.3.2 Initiation of SPR Assay
9.3.3 Micro-calibration (MC)
9.4 Notes
References
10: Optimization and Validation of Strepavidin/Biotin-Based Capture of Ligand
10.1 Introduction
10.2 Materials
10.2.1 30% DMSO Solution (100 mL)
10.2.2 20% DMSO Solution (100 mL)
10.2.3 10% DMSO Solution (100 mL)
10.2.4 10 mM DTT (100 mL)
10.2.5 0.05% Tween20 (100 mL)
10.2.6 3% DMSO in 10 mM HEPES Buffer (10 mL)
10.2.7 2% DMSO in 10 mM HEPES Buffer (1000 mL)
10.2.8 1% DMSO in 10 mM HEPES Buffer (10 mL)
10.2.9 0% DMSO in 10 mM HEPES Buffer (50 mL)
10.2.10 1.5% DMSO in 10 mM HEPES Buffer (10 mL)
10.2.11 2.8% DMSO in 10 mM HEPES Buffer (10 mL)
10.2.12 3% Sucrose in 10 mM HEPES Buffer (10 mL)
10.2.13 Concentrations of Small Molecules Required (Reference)
10.2.14 Preparation of 10 mM Master Stock
10.2.15 Preparation of 0.5 mM Substock
10.2.16 Preparation of 1.25 mM Substock
10.2.17 Preparation of 2.5 mM Substock
10.2.18 Preparation of 10 uM Working Concentration
10.2.19 Preparation of 25 uM Working Concentration
10.2.20 Preparation of 50 uM Working Concentration
10.2.21 SPR Compatible 96 Well Plate
10.3 Methods
10.3.1 Refractive Index (RI) Correction
10.3.2 Initiation of SPR Assay
10.3.3 Micro-calibration (MC)
10.4 Notes
References
11: Preparation of Fragments for Screening in SPR
11.1 Introduction
11.2 Materials
11.2.1 30% DMSO Solution (100 mL)
11.2.2 20% DMSO Solution (100 mL)
11.2.3 10% DMSO Solution (100 mL)
11.2.4 10 mM DTT (100 mL)
11.2.5 0.05% Tween20 (100 mL)
11.2.6 3% DMSO in 10 mM HEPES Buffer (10 mL)
11.2.7 2% DMSO in 10 mM HEPES Buffer (1000 mL)
11.2.8 1% DMSO in 10 mM HEPES Buffer (10 mL)
11.2.9 0% DMSO in 10 mM HEPES Buffer (50 mL)
11.2.10 1.5% DMSO in 10 mM HEPES Buffer (10 mL)
11.2.11 2.8% DMSO in 10 mM HEPES Buffer (10 mL)
11.2.12 3% Sucrose in 10 mM HEPES Buffer (10 mL)
11.3 Methods
11.3.1 Fragment Master Plate (10 mM)
11.3.2 Substock Plate (5 mM)
11.3.3 SPR Assay Plate (100 μM)
11.3.4 Precipitation Check
11.4 Notes
References
12: Screening of Fragments in SPR
12.1 Introduction
12.2 Materials
12.3 Methods
12.3.1 Setup of Protocol for Screening
12.3.2 Initiation of Screening
12.4 Notes
13: Identification of Fragment Hits
13.1 Introduction
13.2 Materials
13.3 Methods
13.4 Notes
Reference
14: Data Analysis and Confirmation of Hits
14.1 Introduction
14.2 Materials
14.3 Methods
14.3.1 Setup of Protocol for Screening
14.3.2 Initiation of Screening
14.3.3 Data Analysis
14.4 Notes
Summary