»
Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate
 
 

Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate, 1st Edition

 
Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate, 1st Edition,Jon Lorsch,ISBN9780124200678
 
 
 

Methods in Enzymology

J Lorsch   

Academic Press

9780124200678

408

229 X 152

In this volume we have brought together a number of core protocols concentrating on Cell, Lipid and Carbohydrate, carefully written and edited by experts.

Print Book

Hardcover

In Stock

Estimated Delivery Time
USD 199.00
 
 

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

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.

Readership

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

Jon Lorsch

Affiliations and Expertise

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

View additional works by Jon Lorsch

Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate, 1st Edition

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

 
 
Free Shipping
Shop with Confidence

Free Shipping around the world
▪ Broad range of products
▪ 30 days return policy
FAQ

Contact Us