chapter  20
24 Pages

Affinity Chromatography in Molecular Biology

Luis A. Jurado, Shilpa Oak, Himanshu Gadgil, Robert A. Moxley, and Harry W. Jarrett Department of Biochemistry, University of Tennessee Health Science Center, Memphis, TN

CONTENTS

20.1 Introduction ......................................................................................................... 548 20.2 Polynucleotide Isolation ...................................................................................... 548

20.2.1 Homopolymer Ligands ......................................................................... 548 20.2.2 Heteropolymer Ligands and Subtractive Hybridization ...................... 549

20.3 Protein Isolation .................................................................................................. 551 20.3.1 Histidine Tags ....................................................................................... 551 20.3.2 Calmodulin-Binding Peptide ................................................................ 553 20.3.3 Strep-Tag............................................................................................... 553 20.3.4 Biotinylation of Fusion Proteins .......................................................... 554 20.3.5 Glutathione S-Transferase..................................................................... 554 20.3.6 Maltose-Binding Protein....................................................................... 555 20.3.7 S Tag Fusion Systems........................................................................... 555 20.3.8 Cellulose-Binding Domain Fusion Proteins......................................... 555

20.4 Work with Immobilized Antibodies and Antigens ............................................. 555 20.4.1 Immunoprecipitation............................................................................. 555 20.4.2 Antibody Binding to Immobilized Bacterial Proteins ......................... 556 20.4.3 Immobilized Antibodies and Antigens ................................................. 558 20.4.4 Cell Isolation......................................................................................... 559

20.5 The ChIP Assay................................................................................................... 560 20.5.1 General Principles of the ChIP Assay.................................................. 560 20.5.2 Experimental Procedures ...................................................................... 562

20.5.2.1 Formaldehyde Fixation of Cells ......................................... 562 20.5.2.2 Chromatin Solubilization .................................................... 562

20.5.2.3 Immunoprecipitation of Cross-Linked Chromatin ............. 563 20.5.2.4 Reversal of Cross-Linked DNA and DNA Purification..... 563 20.5.2.5 Analysis of Immunoprecipitated DNA

and Identification of Binding Sites..................................... 563 20.6 Multiplexed Particle-Based Flow Cytometric Assays ........................................ 564

20.6.1 Nucleic Acid Hybridization.................................................................. 565 20.6.2 Simultaneous Measurement of Cytokines............................................ 566 20.6.3 Analysis of Protein-Protein Interactions .............................................. 566

20.7 Summary and Conclusions.................................................................................. 566 Symbols and Abbreviations ............................................................................................ 567 References ....................................................................................................................... 568

20.1 INTRODUCTION

Affinity chromatography encompasses a variety of techniques and is used in many fields. One of the fields in which it is widely used is molecular biology [1-53]. Although standard columns can be employed in such work, molecular biology often utilizes formats that are uncommon elsewhere. For example, packed columns containing protein A are often used in other fields for antibody purification. However, in molecular biology protein A is instead coupled to microbeads and used for immunoprecipitation. These beaded supports have also been combined with fluorescent dyes and flow cytometry to give a method known as a microsphere-bead assay, which can provide detection limits approaching or exceeding those of high-performance liquid chromatography (HPLC).