Laboratory Methods in Enzymology: Protein Part B

Laboratory Methods in Enzymology: Protein Part B

1st Edition - February 24, 2014
This is the Latest Edition
  • Editor: Jon Lorsch
  • eBook ISBN: 9780124201798
  • Hardcover ISBN: 9780124201200

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Description

Laboratory Methods in Enzymology: Protein Part B brings together a number of core protocols concentrating on protein, carefully written and edited by experts.

Key Features

  • Indispensable tool for the researcher
  • Carefully written and edited by experts to contain step-by-step protocols
  • In this volume we have brought together a number of core protocols concentrating on protein

Readership

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists

Table of Contents

  • Contributors

    Miscellaneous

    Preface

    Section I: Protein Protocols/Protein In Vitro Translation

    Chapter One: In Vitro Synthesis of Proteins in Bacterial Extracts

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Grow and harvest E. coli for the S30 extract

    6 Step 2 Preparation of the S30 extract

    7 Step 3 Optimization of the coupled transcription and translation reaction

    Chapter Two: Preparation of a Saccharomyces cerevisiae Cell-Free Extract for In Vitro Translation

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Preparation of Yeast Cell-Free Extract

    6 Step 2 Cell-Free Translation

    Section II: Protein Protocols/Protein In Vivo Binding Assays

    Chapter Three: Yeast Two-Hybrid Screen

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Small-Scale Transformation of Yeast with pDBLeu-X

    6 Step 2 Two-Hybrid Screen

    7 Step 3 Confirmation of Positive Interactors

    8 Step 4 Plasmid Rescue from Yeast

    9 Step 5 Electroporation of E. coli with Yeast DNA and Identification of Positive Interactors

    10 Step 6 Back-Transformation of Yeast and Further Confirmation of Interactions

    Chapter Four: UV Cross-Linking of Interacting RNA and Protein in Cultured Cells

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 UV Cross-Link RNA–Protein Complexes

    6 Step 2 SDS Lysis of Cells

    7 Step 3 Immunoprecipitation

    8 Step 4 Proteinase K Treatment of RNA Samples

    9 Step 5 RNA Analysis

    10 Step 6 Protein Analysis

    Chapter Five: Analysis of RNA–Protein Interactions by Cell Mixing

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Cell Mixing

    6 Step 2 Cell Lysis

    7 Step 3 Immunoprecipitation

    8 Step 4 Proteinase K Treatment of RNA Samples

    9 Step 5 Northern and Western Blot Analysis

    Chapter Six: General Protein–Protein Cross-Linking

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Calculate the Amount of BS3 to Use

    6 Step 2 Protein Cross-Linking

    Chapter Seven: Chromatin Immunoprecipitation and Multiplex Sequencing (ChIP-Seq) to Identify Global Transcription Factor Binding Sites in the Nematode Caenorhabditis Elegans

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Preparation of Extract from Formaldehyde-Fixed Caenorhabditis elegans Embryos and Larvae

    6 Step 2 Washes and Collection of the Immunocomplexes and ChIP DNA Purification

    7 Step 3 Library Preparation for Multiplex Sequencing Using the Illumina Genome Analyzer

    Acknowledgments

    Chapter Eight: PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 UV Crosslinking of 4-Thiouridine-Labeled Cells (Day 1)

    6 Step 2 Preparation of Cell Lysate for Immunoprecipitation (Day 2)

    7 Step 3 Preparation of the Magnetic Beads (Day 2)

    8 Step 4 Immunoprecipitation and Second RNase T1 Treatment (Day 2)

    9 Step 5 Dephosphorylation and Radiolabeling RNA Segments Crosslinked to Immunoprecipitated Proteins (Day 2)

    10 Step 6 SDS-PAGE and Electroelution of Cross-Linked RNA-Protein Complexes from Gel Slices (Days 2 and 3)

    11 Step 7 Proteinase K Digestion (Day 3)

    12 Step 8 3′-Adapter Ligation for cDNA Library Preparation (Day 3 overnight, day 4, beginning of day 5)

    13 Step 9 5′-Adapter Ligation for cDNA Library Preparation (Day 5, beginning of day 6)

    14 Step 10 cDNA Library Preparation/Reverse Transcription (Day 6)

    15 Step 11 PCR Amplification of cDNA Library & Sample Preparation for Sequencing (Day 6)

    16 Step 12 Determination of Incorporation Levels of 4SU into Total RNA

    Chapter Nine: Determining the RNA Specificity and Targets of RNA-Binding Proteins using a Three-Hybrid System

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Pilot Transformation to Determine Expected Transformation Efficiency

    6 Step 2 Determine 3-AT Concentration to be Used in Selection

    7 Step 3 Introduce the Hybrid RNA Library

    8 Step 4 Assay β-Galactosidase Activity

    9 Step 5 Cure the RNA Plasmid and Test Positives for Protein Dependence

    10 Step 6 Isolate Plasmids for Autoactivation Test and Sequencing

    11 Step 7 Determine Binding Specificity Using Mutant and Control Proteins

    12 Step 8 Functional Tests or Additional Screens

    Chapter Ten: Dissecting a Known RNA–Protein Interaction using a Yeast Three-Hybrid System

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1A Assaying Interactions: Qualitative Filter Assay for β-Galactosidase Activity

    6 Step 1B Assaying Interactions: Quantitative Solution Assay for β-Galactosidase Activity

    7 Step 1C Assaying Interactions: 3-Aminotriazole (3-AT) Resistance Assay

    Chapter Eleven: Identifying Proteins that Bind a Known RNA Sequence Using the Yeast Three-Hybrid System

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Pilot Transformation to Determine Expected Transformation Efficiency

    6 Step 2 Determine 3-AT Concentration to be Used in Selection

    7 Step 3 Introduce the cDNA Library

    8 Step 4 Eliminate RNA-Independent False Positives by Colony Color

    9 Step 5 Assay β-Galactosidase Activity

    10 Step 6 Cure the RNA Plasmid and Test Positives for RNA Dependence

    11 Step 7 Isolate Plasmids for Autoactivation Test and Sequencing

    12 Step 8 Determine Binding Specificity Using Mutant and Control RNAs

    13 Step 9 Functional Tests or Additional Screens

    Author Index

    Subject Index

Product details

  • No. of pages: 240
  • Language: English
  • Copyright: © Academic Press 2014
  • Published: February 24, 2014
  • Imprint: Academic Press
  • eBook ISBN: 9780124201798
  • Hardcover ISBN: 9780124201200

About the Serial Volume Editor

Jon Lorsch

Affiliations and Expertise

Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA