Laboratory Methods in Enzymology Protein Part B 1st Edition by Jon Lorsch ISBN 0124201202 9780124201200

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ISBN 10: 0124201202 
ISBN 13: 9780124201200
Author: Jon Lorsch

Laboratory Methods in Enzymology Protein Part B 1st Table of contents:

Section I: Protein Protocols / Protein In Vitro Translation

Chapter One: In Vitro Synthesis of Proteins in Bacterial Extracts

• Abstract

• 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

• Abstract

• 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

• Abstract

• 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

• Abstract

• 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

• Abstract

• 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

• Abstract

• 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 Caenorhabditis elegans

• Abstract

• 1 Theory

• 2 Equipment

• 3 Materials

• 4 Protocol

• 5 Step 1: Preparation of Extract from Formaldehyde-Fixed C. elegans Embryos and Larvae

• 6 Step 2: Washes and Collection of 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

• Abstract

• 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 Magnetic Beads (Day 2)

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

• 9 Step 5: Dephosphorylation and Radiolabeling of 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

• Abstract

• 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

• Abstract

• 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

• Abstract

• 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

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Tags: Jon Lorsch, Laboratory Methods, Enzymology Protein