Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate
- 1st Edition, Volume 533 - November 12, 2013
- Latest edition
- Editor: Jon Lorsch
- Language: English
- Hardback ISBN:9 7 8 - 0 - 1 2 - 4 2 0 0 6 7 - 8
- eBook ISBN:9 7 8 - 0 - 1 2 - 4 2 0 0 9 4 - 4
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-… Read more
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.
- 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
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
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
- Edition: 1
- Latest edition
- Volume: 533
- Published: November 12, 2013
- Language: English
JL
Jon Lorsch
Affiliations and expertise
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USARead Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate on ScienceDirect