ABSTRACT
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).
