Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate

Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate

1st Edition - September 24, 2013
This is the Latest Edition
  • Editor: Jon Lorsch
  • eBook ISBN: 9780124200944

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Description

Methods in Enzymology volumes provide an indispensable tool for the researcher. Each volume is carefully written and edited by experts to contain state-of-the-art reviews and step-by-step protocols. In this volume, we have brought together a number of core protocols concentrating on Cell, Lipid and Carbohydrate, complementing the traditional content that is found in past, present and future Methods in Enzymology volumes.

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 Cell, Lipid and Carbohydrate

Readership

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

Table of Contents

  • Series Page

    Contributors

    Miscellaneous

    Preface

    Methods in Enzymology

    Section I: Cell Protocols: Basic Microbiological Techniques

    Chapter One. Pouring Agar Plates and Streaking or Spreading to Isolate Individual Colonies

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Preparation of LB-Agar Solution

    6 Step 2 Pouring Agar Plates

    7 Step 3 Streaking for Individual Colonies

    8 Step 4 Spreading Cells on Agar Plates

    References

    Chapter Two. Storage of Bacteria and Yeast

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Protocol A Cryopreservation of Bacterial Cultures

    6 Step 1A Grow Bacterial Cultures

    7 Step 2A Freeze the Bacterial Cells

    8 Protocol B Cryopreservation of Yeast

    9 Step 1B Grow Yeast Cultures

    10 Step 2B Freeze the Yeast Cells

    References

    Section II: Cell Protocols: Cellular Fractionation

    Chapter Three. Cellular Fractionation – Mammalian Cells

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Subcellular Mammalian Cell Fractionation

    Source References

    Chapter Four. Cellular Fractionation – Yeast Cells

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Yeast Protoplast Formation

    6 Step 2 Subcellular Fractionation of Yeast Protoplasts

    Source References

    Section III: Cell Protocols: Knock-outs and Gene

    Chapter Five. Yeast-Gene Replacement Using PCR Products

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Generation of a Linear Integrating Construct by PCR

    6 Step 2 Visualization of Product on an Agarose Gel

    7 Step 3 Transforming Yeast with a Linear PCR Product for Integration

    8 Step 4 Verifying Appropriate Integration in Colonies Growing on Selective Media

    Source References

    Chapter Six. Measuring RNAi Knockdown using qPCR

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Transfection

    6 Step 2 RNA Isolation Using MagMAX™ Magnetic Bead Purification

    7 Step 3 cDNA Synthesis

    8 Step 4 qPCR

    9 Step 5 qPCR Analysis

    References

    Referenced Protocols in Methods Navigator

    Chapter Seven. Recombineering: Using Drug Cassettes to Knock out Genes in vivo

    Abstact

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Design Construct and Order Primers

    6 Step 2 Set Up and Generate Linear Substrate by PCR

    7 Step 3 Preparing Cells Competent for Recombineering

    8 Step 4 Electrotransformation of Linear Substrates into the Recombineering-Ready Cells

    9 Step 5 Selecting for Knockout Mutations

    10 Step 6 Confirming Knockout Mutations

    References

    Related Literature

    Referenced Protocols in Methods Navigator

    Chapter Eight. Gene Knockouts, in vivo Site-Directed Mutagenesis and Other Modifications Using the Delitto Perfetto System in Saccharomyces cerevisiae

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 PCR Amplification of the CORE Cassette

    6 Step 2 Agarose Gel Electrophoresis to Visualize the Product of PCR

    7 Step 3 Concentration of the PCR Product

    8 Step 4 Insertion of the CORE Cassette into the Chosen Genetic Locus

    9 Step 5 Colony PCR of the Transformants

    10 Step 6 Transformation Using DNA Oligonucleotides to Generate the Desired mutation(s)

    References

    Source References

    Chapter Nine. Gene Targeting and Site-Specific Recombination in Mouse ES Cells

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Delivery of Targeting Vector into ES Cells by DNA Transfection

    6 Step 2 Picking and Expansion of Colonies

    7 Step 3 Isolation of DNA from 96-Well Plates

    8 Step 4 Recombinase Delivery into ES Cells by DNA Transfection

    9 Step 5 Screen the Colonies for Complete Recombination and Removal of the Selection Cassette

    References

    Source References

    Chapter Ten. Recombineering: Highly Efficient in vivo Genetic Engineering using Single-strand Oligos

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Designing the Oligo for Recombineering

    6 Step 2 Preparing Cells Competent for Recombineering

    7 Step 3 Electrotransformation of the Oligo into the Recombineering-ready Cells

    8 Step 4 Plating Cells to Screen for Mutations

    9 Step 5 Screening for the Final Construct

    References

    Related Literature

    Referenced Protocols in Methods Navigator

    Section IV: Cell Protocols: Microbiological Media

    Chapter Eleven. Growth Media for E. coli

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Recipe 1 LB

    5 Recipe 2 TB

    6 Recipe 3 2× YT

    7 Recipe 4 SOC

    References

    Chapter Twelve. Saccharomyces Cerevisiae Growth Media

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Recipe 1 Preparation of YPD

    6 Recipe 2 Preparation of Synthetic Complete (Drop-out) Media

    7 Recipe 3 Preparation of Minimal Media

    8 Recipe 4 Preparation of Sporulation Media

    References

    Section V: Cell Protocols: Preparation of Slides for Microscopy

    Chapter Thirteen. Immunohistochemistry on Freely Floating Fixed Tissue Sections

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Pretreatment of Tissue Section

    6 Step 2 Blocking Nonspecific Binding

    7 Step 3 Primary Antibody Labeling

    8 Step 4 Secondary Antibody Labeling

    9 Step 5 Mount Labeled Tissue Section on Microscope Slide

    Chapter Fourteen. Preparation of Slides for Microscopy from Frozen Tissue Sections

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1A Submerging Cut-Tissue Sections in Cryoprotectant Media

    6 Step 1B Embedding Tissue Samples

    7 Step 2 Cryostat Sectioning

    8 Step 3 Preparation of Slides for Immunochemistry

    References

    Chapter Fifteen. Preparation of Formalin-fixed Paraffin-embedded Tissue for Immunohistochemistry

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Fixation of Tissue

    6 Step 2 Tissue Processing and Embedding

    7 Step 3 Tissue Sectioning

    References

    Chapter Sixteen. Preparation of Cells for Microscopy using Cytospin

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Collecting Cells

    6 Step 2 Cytospin

    7 Step 3 Fixation and Drying

    References

    Chapter Seventeen. Preparation of Cells for Microscopy using Chamber Slides and Coverslips

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Collecting & Seeding Cells

    6 Step 2 Treatment of Cells

    7 Step 3 Fixation of Cells

    References

    Chapter Eighteen. Preparation of Cells for Microscopy using ‘Cell Blocks’

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Collection and Fixation of Cells

    6 Step 2 Making Cell Plugs

    7 Step 3 Paraffin Embedding and Sectioning

    References

    Section VI: Lipid and Carbohydrate Protocols

    Chapter Nineteen. Enzymatic Deglycosylation of Glycoproteins

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Enzymatic Deglycosylation of Glycoprotein

    References

    Source References

    Section VII: Lipid and Carbohydrate Protocol: Making Vesicles and Micelles

    Chapter Twenty. Preparation of Fatty Acid or Phospholipid Vesicles by Thin-film Rehydration

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Formation of a Thin Lipid Film

    6 Step 2 Rehydration of the Thin Lipid Film

    References

    Source References

    Chapter Twenty-one. Vesicle Extrusion Through Polycarbonate Track-etched Membranes using a Hand-held Mini-extruder

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Assemble the Extrusion Apparatus

    6 Step 2 Load the Gas-Tight Syringes

    7 Step 3 Extrusion to Yield Monodisperse Vesicles

    References

    Related Literature

    Referenced Protocols in Methods Navigator

    Chapter Twenty-two. Preparation of Fatty Acid Micelles

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Dissolve Fatty Acid in NaOH Solution

    6 Step 2 Addition of Fatty Acid Micelles to Vesicles

    References

    Source References

    Section VIII: Other Protocols

    Chapter Twenty-three. Reverse-phase HPLC Analysis and Purification of Small Molecules

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Break Down the Matrix of the Samples that Contain the Molecule of Interest

    6 Step 2 Extraction of Carotenoids from the Samples

    7 Step 3 Carotenoid Analysis on a Reverse-Phase HPLC-PDA System

    References

    Source References

    Chapter Twenty-Four. Thin Layer Chromatography

    Abstact

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Preparing the TLC Plates

    6 Step 2 Sample Application

    7 Step 3 Development of the Plate

    8 Step 4 Detection and Visualization of the Sample

    References

    Referenced Protocols in Methods Navigator

    Chapter Twenty-five. ATP and GTP Hydrolysis Assays (TLC)

    Abstract

    1 Theory

    2 Equipment

    3 Materials

    4 Protocol

    5 Step 1 Steady-state ATP Hydrolysis

    6 Step 2 Preparation of the TLC Plate and Chamber

    7 Step 3 TLC Separation of the Reaction

    References

    Author Index

    Subject Index

Product details

  • No. of pages: 408
  • Language: English
  • Copyright: © Academic Press 2013
  • Published: September 24, 2013
  • Imprint: Academic Press
  • eBook ISBN: 9780124200944

About the Serial Editor

Jon Lorsch

Affiliations and Expertise

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