Basic Protocol: Labeling of DNA by Random Oligonucleotide-Primed
Synthesis 3.5.9

T4 DNA Polymerase 3.5.11

Native T7 DNA Polymerase 3.5.12

Modified T7 DNA Polymerase 3.5.13

Taq DNA Polymerase 3.5.14

Literature Cited 3.5.15

Template-Independent DNA Polymcrascs 3.6.1

Terminal Deoxynucleotidyltransferase (Terminal Transferase) 3.6.1

RNA-Dependent DNA Polymerascs 3.7.1

Reverse Transcriptase 3.7.1

DNA-Dcpcndcnt RNA Polymerases 3.8.1

E. coli RNA Polymerase 3 .8. l

Phage RNA Polymerases: SP6, T7, T3 3.8.2

DNA-lndcpcndcnt RNA Polymerases 3.9.1

Poly(A) Polymerase 3.9. l

Phosphatascs and Kinases 3.10.1

Alkaline Phosphatases: Bacterial Alkaline Phosphatase and Calf Intestine
Phosphatase 3.10.1

T4 Polynucleotide Kinase 3.10.2

Basic Protocol: Labeling 5' Ends by the Forward Reaction 3.10.3

Basic Protocol: Phosphorylating Synthetic Oligonucleotides by the Forward
Reaction 3.10.4

Basic Protocol: Labeling 5' Termini by the Exchange Reaction 3.10.4

Exonucicascs 3.11.1

Single-Stranded 5'-3' and 3'-5' Exonucleases 3.11.1

Double-Stranded 5'-3' Exonucleases 3.11.2

Double-Stranded 3'-->5' Exonucleases 3.11.3

Endonucleascs 3.12.1

Bal 31 Nuclease 3.12.1

S1 Nuclease 3.12.2

Mung Bean Nuclease 3.12.3

Micrococcal Nuclease 3.12.4

Deoxyribonuclease I (DNase I) 3.12.5

Ribomicleascs 3.13.1

RibonucleaseA 3.13.1

RibonucleaseH 3.13.2

RibonucleaseTI 3.13.2


DNA Ligascs 3.14.1

T4DNALigase 3.14.1

E. coli DNALigase 3.14.3

RNA Ligascs 3.15.1

T4RNALigase 3.15.1

CONSTRUCTION OF HYBRID DNA MOLECULES

Subcloning of DNA Fragments 3.16.1

Basic Protocol 3.16.1

Alternate Protocol: Ligation of DNA Fragments in Gel Slices 3.16.3

Reagents and Solutions 3.16.3

Commentary 3.16.4

Example: Subcloning DNA Fragments with Homologous and Cohesive Ends 3.16.7

Example: Directional Cloning Using Fragments with Heterologous Ends 3.16.8

Example: Blunt-End Ligation 3.16.8

Example: Joining DNA Fragments with Incompatible Ends 3.16.8

Example: Ligation of Fragments Generated by Partial Cleavage 3.16.9

Example: Ligations Involving Oligonucleotide Linkers 3.16.9

Example: Cloning Synthetic Oligonucleotides 3.16.10

Constructing Recombinant DNA Molecules by the
Polymerase Chain Reaction 3.17.1

Basic Protocol: Subcloning DNA Fragments 3.17.1

Commentary 3.17.3

SPECIALIZED APPLICATIONS

Labeling and Colorimetric Detection of Nonisotopic Probes 3.18.1

Basic Protocol: Preparation of Biotinylated Probes by Nick Translation 3.18.1

Basic Protocol: Preparation of Biotinylated Probes by Random
Oligonucleotide-Primed Synthesis 3.18.3

Support Protocol: Colorimetric Detection of Biotinylated Probes 3.18.4

Alternate Protocol: Preparation and Detection of Digoxigenin-Labeled
DNA Probes 3.18.5

Reagents and Solutions 3.18.6

Commentary 3.18.7

Chemiluminescent Detection of Nonisotopic Probes 3.19.1

Basic Protocol: Chemiluminescent Detection of Biotinylated Probes 3.19.1

Alternate Protocol: Chemiluminescent Detection of Digoxigenin-Labeled
Probes 3.19.4

Support Protocol: Calibrating an Ultraviolet Light Source 3.19.5

Reagents and Solutions 3.19.5

Commentary 3.19.6
PREPARATION AND
ANALYSIS OF RNA

INTRODUCTION 4.0.3

PREPARATION OF RNA FROM EUKARYOTIC AND
PROKARYOTIC CELLS

Preparation of Cytoplasmic RNA from Tissue Culture Cells 4.1.2

Basic Protocol 4.1.2

Short Protocol: Preparation of Cytoplasmic RNA from Tissue Culture Cells 4.1.4

Support Protocol: Removal of Contaminating DNA 4.1.4

Reagents and Solutions 4.1.5

Commentary 4.1.5

Guanädine Methods for Total RNA Preparation 4.2.1

Basic Protocol: Single-Step RNA Isolation from Cultured Cells or Tissues 4.2.1

Alternate Protocol I: CsCI Purification of RNA from Cultured Cells 4.2.3

Alternate Protocol 2: CsCI Purification of RNA from Tissue 4.2.5

Reagents and Solutions 4.2.6

Commentary 4.2.7

Phenol/SDS Method for Plant RNA Preparation 4.3.1

Basic Protocol 4.3.1

Reagents and Solutions 4.3.3

Commentary 4.3.3

Preparation of Bacterial RNA 4.4.1

Basic Protocol: Isolation of High-Quality RNA ftom Gram-Negative Bacteria 4.4.1

Basic Protocol: Isolation of RNA from Gram-Positive Bacteria 4.4.3

Alternate Protocol: Rapid Isolation of RNA from Gram-Negative Bacteria 4.4.4

Reagents and Solutions 4.4.5

Commentary 4.4.6

Preparation of Poly(A)+ RNA 4.5.1

Basic Protocol 4.5.1

Reagents and Solutions 4.5.2

Commentary 4.5.2

ANALYSIS OF RNA STRUCTURE AND SYNTHESIS

S 1 Analysis of Messenger RNA Using Single-Stranded DNA Probes 4.6.1

Basic Protocol: SI Analysis of mRNA Using M13 Template 4.6.2

Short Protocol: Preparation of Single-Stranded End-Labeled Probe
and S I Analysis of mRNA 4.6.6

Alternate Protocol: Synthesis of Single-Stranded Probe from
Double-Stranded Plasmid Template 4.6.7

Alternate Protocol: Quantitative SI Analysis of mRNA Using
Oligonucleotide Probes 4.6.7

Support Protocol: Controls for Quantitative S I Analysis of mRNA 4.6.10

Reagents and Solutions 4.6.10

Commentary 4.6.11

Ribonucicase Protection Assay 4.7.1

Basic Protocol 4.7.1

Support Protocol: Gel Purification of RNA Probes 4.7.3

Support Protocol: Preparation of Template DNA 4.7.4

Reagents and Solutions 4.7.5

Commentary 4.7.6

Primer Extension 4.8.1

Basic Protocol 4.8.1

Reagents and Solutions 4.8.3

Commentary 4.8.4

Analysis of RNA by Northern and Slot Blot Hybridization 4.9.1

Basic Protocol: Northern Hybridization of RNA Fractionated by
Agarose-Formaldehyde Gel Electrophoresis 4.9.2

Alternate Protocol I: Northern Hybridization of RNA Denatured by
Glyoxal/DMSO Treatment 4.9.8

Alternate Protocol 2: Northern Hybridization of Unfractionated RNA
Immobilized by Slot Blotting 4.9.9

Support Protocol: Removal of Probes from Northern Blots 4.9.11

Reagents and Solutions 4.9.12

Commentary 4.9.13

Identification of Newly Transcribed RNA 4.10.1

Basic Protocol: Nuclear Runoff Transcription in Mammalian Cells 4.10.1

Alternate Protocol: Isolation of Nuclei by Dounce Homogenization 4.10.4

Alternate Protocol: Isolation of Nuclei by Sucrose Gradient Centrifugation 4.10.6

Support Protocol: Preparation ofNitrocellulose Filters for Nuclear Runoff
Transcription Assay 4.10.7

Reagents and Solutions 4.10.8

Commentary 4.10.10
CONSTRUCTION OF
RECOMBINANT DNA
LIBRARIES

INTRODUCTION 5.0.3

OVERVIEW OF RECOMBINANT DNA LIBRARIES

Genomic DNA Libraries 5.1.1

Representation and Randomness 5.1.1

Subgenomic Libraries 5.1.2

Vectors for Genomic DNA Libraries 5.1.2

cDNA Libraries 5.2.1

PREPARATION OF INSERT DNA FROM GENOMIC DNA

Size Fractionation Using Sucrose Gradients 5.3.2

Basic Protocol: Sucrose Gradient Preparation of Size-Selected DNA 5.3.2

Support Protocol: Partial Enzyme Digestion 5.3.4

Support Protocol: Complete Enzyme Digestion 5.3.5

Reagents and Solutions 5.3.6

Commentary 5.3.6

Size Fractionation Using Agarose Gels 5.4.1

Basic Protocol: Electrophoresis on a Slab Agarose Gel 5.4.1

Alternate Protocol: Electrophoresis on the Builds-Eye Agarose
Gel Apparatus 5.4.1

Commentary 5.4.3

PREPARATION OF INSERT DNA FROM MESSENGER RNA

Conversion of mRNA into Double-Stranded cDNA 5.5.2

Basic Protocol: Conversion of mRNA into Blunt-Ended Double-
Stranded cDNA 5.5.2

Alternate Protocol: Conversion of mRNA into Double-Stranded cDNA
for Directional Cloning 5.5.6

Reagents and Solutions 5.5.10

Commentary 5.5.12

Ligation of Linkers or Adapters to Double-Stranded cDNA 5.6.1

Basic Protocol: Methylation of cDNA and Ligation of Linkers 5.6.1

Alternate Protocol: Ligation of BstXl Synthetic Adapters 5.6.4

Support Protocol: Preparation of a CL-4B Column 5.6.5

Reagents and Solutions 5.6.7

Commentary 5.6.8

IV PRODUCTION OF GENOMIC DNA AND cDNA LIBRARIES

Production of a Genomic DNA Library 5.7.1

Basic Protocol 5.7.1

Commentary 5.7.3

SCREENING OF RECOMBINANT
DNA LIBRARIES

INTRODUCTION 6.0.3

PLATING LIBRARIES AND TRANSFER TO FILTER MEMBRANES

Plating and Transferring Bacteriophage Libraries 6.1.1

Basic Protocol 6.1.1

Commentary 6.1.3

Plating and Transferring Cosmid and Plasmid Libraries 6.2.1

Basic Protocol 6.2.1

Commentary 6.2.3

HYBRIDIZATION WITH RADIOACTIVE PROBES

Using DNA Fragments as Probes 6.3.1

Basic Protocol: Hybridization in Formamide 6.3.1

Alternate Protocol: Hybridization in Aqueous Solution 6.3.3

Reagents and Solutions 6.3.4

Commentary 6.3.5

Using Synthetic Oligonucleotides as Probes 6.4.1

Basic Protocol: Hybridization in Sodium Chloride/Sodium Citrate (SSC) 6.4.1

Basic Protocol: Hybridization in Tetramethylammonium Chloride (TMAC) 6.4.3

Support Protocol: Labeling the 5' Ends of Mixed Oligonucleotides 6.4.6

Reagents and Solutions 6.4.6

Commentary 6.4.7

PURIFICATION OF BACTERIOPHAGE, COSMID,
AND PLASMID CLONES

Purification of Bacteriophage Clones 6.5.1

Basic Protocol 6.5.1

Commentary 6.5.2

Purification of Cosmid and Plasmid Clones 6.6.1

Basic Protocol 6.6.1

Commentary 6.6.1

SCREENING WITH ANTIBODIES

Immunoscreening of Fusion Proteins Produced in Lambda Plaques 6.7.1

Basic Protocol: Screening a (lambdal)gt 11 Expression Library with Antibodies 6.7.1

Short Protocol: Screening a (lambdal) gt 11 Expression Library with Antibodies 6.7.3

Alternate Protocol: Induction of Fusion Protein Expression with IPTG
prior to Screening with Antibodies 6.7.3

Commentary 6.7.4

Immunoscreening after Hybrid Selection and Translation 6.8.1

Basic Protocol - 6.8.1

Reagents and Solutions 6.8.4

Commentary 6.8.4

YEAST ARTIFICIAL CHROMOSOME LIBRARIES

Overview of Strategies for Screening YAC Libraries and
Analyzing YAC Clones 6.9.1

Generating YAC Libraries 6.9.1

YAC Library Screening by a Core Laboratory 6.9.2

Designing a Locus-Specific PCR Assay for Screening 6.9.2

Fragment size 6.9.4

Primer length 6.9.4

Primer affinity 6.9.4

Analyzing Individual YAC Clones 6.9.4

Chimerism of the YAC Insert 6.9.4

Internal Rearrangement or Instability of the YAC Insert 6.9.5

Construction and Analysis of a YAC-lnsert Sublibrary 6.9.6

Analysis of Isolated YAC Clones 6.10.1

Basic Protocol: Propagation and Storage of YAC-Containing Yeast Strains 6.10.1

Basic Protocol: Preparation of YAC-Containing DNA from Yeast Clones
for Analysis by Southern Blotting 6.10.2

Basic Protocol: Preparation of Yeast Chromosomes in Agarose Plugs for
Pulsed- Field Gel Electrophoresis 6.10.4

Basic Protocol: End-Fragment Analysis Using PCR Amplification 6.10.6

Alternate Protocol: End-Fragment Analysis by Subcloning into a Bacterial
Plasmid Vector 6.10.9

Support Protocol: Design and Preparation ofpUC19-ES and pUC19-HS
Subcloning Vector 6.10.11

Basic Protocol: Preparation of High-Molecular-Weight YAC-Containing
Yeast DNA insolution 6.10.13

Basic Protocol: Preparation and Analysis of a YAC-lnsert Sublibrary 6.10.14

Reagents and Solutions 6.10.16

Commentary 6.10.17


SPECIALIZED STRATEGIES FOR SCREENING LIBRARIES

Use of Monoclonal Antibodies for Expression Cloning 6.11.1

Basic Protocol: Isolation of cDNA Clones Encoding Cell-Surface Antigens 6.11.1

Support Protocol: Preparation of Antibody-Coated Plates 6.11.6

Basic Protocol: Isolation of cDNA Clones Encoding Intracellular Antigens 6.11.7

Support Protocol: Preparation ofPolyvinylidene-Wrapped Plates 6.11.10

Commentary 6.11.11


Recombination-Based Assay (RBA) for Screening Bacteriophage

Lambda Libraries 6.12.1

Basic Protocol 6.12.1

Reagents and Solutions 6.12.6

Commentary 6.12.7
DNA SEQUENCING

INTRODUCTION 7.0.3

Dideoxy (Sänger) Sequencing 7.0.3

Chemical (Maxam-Gilbert) Sequencing 7.0.7

Choosing Between Dideoxy and Chemical Sequencing Methods 7.0.7

Alternatives to Radiolabeled Sequencing Reactions 7.0.9

Developments in Sequencing Technology 7.0.9

Computer Analysis 7.0.10

Literature Cited 7.0.11

DNA Sequencing Strategies 7.1.1

Dideoxy Sequencing 7.1.1

Chemical Sequencing 7.1.5

Constructing Nested Deletions for Use in DNA Sequencing 7.2.1

Basic Protocol: Using Exonuclease IH to Construct Unidirectional Deletions 7.2.1

Support Protocol: Protection of DNA from Exonuclease III Digestion
Using (alfa S)dNTPs 7.2.8

Basic Protocol: Using Bal 31 Exonuclease to Construct Nested Deletions 7.2.8

Support Protocol: Preparation ofM13mp Sequencing Vector DNA for
Subcloning of Bal 31 -Digested DNA Fragments 7.2.17

Reagents and Solutions 7.2.18

Commentary 7.2.18

Preparation of Templates for DNA Sequencing 7.3.1

Basic Protocol: Preparation of Single-Stranded M13 Phage DNA 7.3.1

Basic Protocol: Preparation of (lambda) DNA from Small-Scale Lysates 7 .3.3

Basic Protocol: Miniprep ofRecombinant pSP64CS orpSP65CS Plasmid
DNA for Chemical Sequencing 7.3.5

Basic Protocol: Miniprep of Double-Stranded Plasmid DNA for Dideoxy
Sequencing 7.3.6

Basic Protocol: Alkali Denaturation of Double-Stranded Plasmid DNA
for Dideoxy Sequencing 7.3.7

Basic Protocol: Preparation of Plasmid DNA from an E. Coli Colony or
Phage DNA from a Plaque for Thermal Cycle Sequencing 7.3.8

Reagents and Solutions 7.3.9

Commentary 7.3.9

DNA Sequencing by the Dideoxy Method 7.4.1

Basic Protocol: Labeling/Termination Sequencing Reactions Using Sequenase 7.4.2

Alternate Protocol: Using Mn+ + in the Labeling/Termination Reactions 7.4.4

Alternate Protocols: Using Other Polymerases in the Labeling/Termination
Reactions 7.4.5

Labeling/Termination Reactions Using Taq DNA Polymerase 7.4.5

Labeling/Termination Reactions Using Klenow Fragment 7.4.5

Basic Protocol: Sequencing by the Sänger Procedure Using Klenow Fragment 7.4.6

Alternate Protocol: Using Taq DNA Polymerase in the Sänger Procedure 7.4.7

Alternate Protocol: One-Step Sequencing Reactions Using 5'-End-
Labeled Primers 7.4.7

Basic Protocol: Thermal Cycle Sequencing Reactions Using (alfa)-Labeled
Nucleotides 7.4.9

Alternate Protocol: Thermal Cycle Sequencing Reactions Using
5'-End-Labeled Primers 7.4.11

Reagents and Solutions 7.4.12

Commentary 7.4.16

Didcoxy DNA Sequencing with Chemiluminescent Detection 7.4.36

Basic Protocol: DNA Sequencing Using Biotinylated Primers with
Chemiluminescent Detection 7.4.36

Alternate Protocol: Two-Step (Indirect) Detection Using Streptavidin and
Biontinylated Alkaline Phosphatase 7.4.40

Alternate Protocol: Sequencing with Hapten-Labeled Primers and
Detection with Antibody-Alkaline Phosphatase Conjugates 7.4.41

Reagents and Solutions 7.4.42

Commentary 7.4.44

DNA Sequencing by the Chemical Method 7.5.1

Basic Protocol: Chemical Sequencing using (23)P-Labeled DNA 7.5.2

Support Protocol: Tth 111 I Digestion and End Labeling 7.5.4

Reagents and Solutions 7.5.7

Commentary 7.5.7


Denaturing Gel Electrophorcsis for Sequencing 7.6.1

Basic Protocol: Pouring, Running, and Processing Sequencing Gels 7.6.1

Alternate Protocol: Buffer-Gradient Sequencing Gels 7.6.7

Alternate Protocol: Electrolyte-Gradient Sequencing Gels 7.6.8

Alternate Protocol: Formamide-Containing Sequencing Gels 7.6.9

Reagents and Solutions 7.6.9

Commentary 7.6.11


Computer Manipulation of DNA and Protein Sequences 7.7.1


Sequence Data Entry 7.7.1


Sequence Data Verification 7.7.5

Restriction Mapping 7.7.7

Prediction of Nucleic Acid Structure 7.7.8

Oligonucleotide Design Strategy 7.7.9

Identification of Protein-Coding Regions 7.7.11


Homology Searching 7.7.12

Genetic Sequence Databases and Other Electronic Resources Available to
Molecular Biologists 7.7.15

Literature Cited 7.7.16

Appendix 7.7.17
MUTAGENESIS OF
CLONED DNA

INTRODUCTION 8.0.3

OHgonucleotidc-Dircctcd Mutagenesis without Phenotypic Selection 8.1.1

Basic Protocol 8.1.1

Reagents and Solutions 8.1.5

Commentary 8.1.5

Mutagenesis with Degenerate Oligonucleotides: Creating Numerous
Mutations in a Small DNA Sequence 8.2.1

Basic Protocol 8.2.1

Commentary 8.2.5

Gene Synthesis: Assembly of Target Sequences Using Mutually
Priming Long Oligonucleotides 8.2.8

Basic Protocol 8.2.8

Commentary 8.2.10

Region-Specific Mutagenesis 8.3.1

Basic Protocol 8.3.1

Support Protocol: Enrichment of Mutant Clones 8.3.4

Commentary 8.3.5

Linker-Scanning Mutagenesis of DNA 8.4.1

Basic Protocol: Linker Scanning Using Nested Deletions and
Complementary Oligonucleotides 8.4.2

Alternate Protocol: Linker Scanning Using Oligonucleotide-Directed
Mutagenesis 8.4.5

Commentary 8.4.6


Directed Mutagenesis Using the Polymerase Chain Reaction 8.5.1

Basic Protocol I: Introduction of Restriction Endonuclease Sites
byPCR 8.5.1

Basic Protocol 2: Introduction of Point Mutations by PCR 8.5.5

Alternate Protocol: Introduction of a Point Mutation by Sequential
PCR Steps 8.5.7

Commentary 8.5.9

INTRODUCTION OF DNA
INTO MAMMALIAN CELLS

INTRODUCTION 9.0.1

TRANSFECTION OF DNA INTO EUKARYOTIC CELLS

Calcium Phosphate TYansfection 9.1.4

Basic Protocol: Transfection Using Calcium Phosphate-DNA Precipitate
Formed in HEPES 9.1.4

Support Protocol: Glycerol/DMSO Shock of Mammalian Cells 9.1.7

Alternate Protocol: High-Efficiency Transfection Using Calcium
Phosphate-DNA Precipitate Formed in B ES 9.1.7

Reagents and Solutions 9.1.8

Commentary 9.1.9

Transfection Using DEAE-Dextran 9.2.1

Basic Protocol: General Procedure for DEAE-Dextran Transfection 9.2.1

Alternate Protocol 1: Sample Experiment: Transfection to Test
Promoter Function 9.2.4

Alternate Protocol 2: Sample Experiment: Transfection to Test
Enzyme Structure/Activity Relationships 9.2.6

Support Protocol: Charcoal Stripping of Fetal Bovine Serum 9.2.7

Reagents and Solutions 9.2.8

Commentary 9.2.8

Transfection by Electroporation 9.3.1

Basic Protocol: Electroporation into Mammalian Cells 9.3.1

Alternate Protocol: Electroporation into Plant Protoplasts 9.3.2

Reagents and Solutions 9.3.3

Commentary 9.3.3

Liposome-MediatedlYansfection 9.4.1

Basic Protocol: Transient Expression Using Liposomes 9.4.1

Alternate Protocol: Stable Transformation Using Liposomes 9.4.2

Reagents and Solutions 9.4.2

Commentary 9.4.3


Selection of Transfected Mammalian Cells 9.5.1

Strategic Planning 9.5.1

Basic Protocol I: Stable Transfer of Genes into Mammalian Cells 9.5.4

Basic Protocol 2: Selectable Markers for Mammalian Cells 9.5.6

Basic Protocol 3: Rapid Selection ofTransfected Mammalian Cells 9.5.10

Support Protocol: Optimization of Cotransfection Conditions 9.5.12

Reagents and Solutions 9.5.13

Commentary 9.5.13


USES OF FUSION GENES IN MAMMALIAN TRANSFECTION

Overview of Genetic Reporter Systems 9.6.3

Design of Reporter Vectors 9.6.4

In Vitro Reporter Assays 9.6.5

In Vivo Reporter Assays 9.6.8

Literature Cited 9.6.11


Isotopic Assay for Reporter Gene Activity 9.7.1

Basic Protocol I: Chromatographie Assay for CAT Activity 9.7.1

Alternate Protocol I: In Situ Lysis of Cells for CAT Assays 9.7.4

Alternate Protocol 2: Phase-Extraction Assay for CAT Activity 9.7.5

Basic Protocol 2: Radioirnmunoassay for Human Growth Hormone 9.7.6

Reagents and Solutions 9.7.8

Commentary 9.7.8

Nonisotopic Assays for Reporter Gene Activity 9.7.12

Basic Protocol I: Firefly Luciferase Reporter Gene Assay 9.7.12

Alternate Protocol: Luciferase Assay in Freeze-Thaw-Lysed Cells 9.7.14

Basic Protocol 2: Chemiluminescent P-Galactosidase Reporter
Gene Assay 9.7.15

Reagents and Solutions 9.7.17

Commentary 9.7.18

Use of the A. Victoria Green Fluorescent Protein to Study Protein
Dynamics in Vivo 9.7.22

Overview of GFP Fluorescence 9.7.22

Utilization of GFP 9.7.22

Problems with GFP 9.7.24

MutantsofGFP 9.7.25

Microscopy Setup 9.7.26


Conclusions 9.7.27

Literature Cited 9.7.27


Direct Analysis of RNA After Transfection 9.8.1

Transfection Efficiency 9.8.1

RNA Preparation 9.8.1

Analysis of RNA 9.8.1

Promoter Strength 9.8.2

TRANSDUCTION OF GENES USING RETROVIRUS VECTORS

Overview of the Retrovirus Transduction System 9.9.1

Retrovirus Life Cycle 9.9.1

Replication-Incompetent Vectors 9.9.4

Replication-Competent Vectors 9.9.6

Packaging Lines and Virus Production 9.9.6

Murine Retroviruses 9.9.11

Avian Retroviruses 9.9.14

Safety Issues 9.9.14

Literature Cited 9.9.14

Preparation of a Specific Retrovirus Producer Cell Line 9.10.1

Basic Protocol I: Introduction of a Retrovirus Vector into a
Packaging Cell Line 9.10.1

Basic Protocol 2: Determination of Viral Titer: Identification of
Producer Clones Making High-Titer Virus 9.10.5

Support Protocol I: Sample Calculation of Titer for Bag Virus 9.10.8

Alternate Protocol: Rapid Evaluation of Producer Colonies 9.10.9

Support Protocol 2: Xgal Staining of Cultured Cells 9.10.9

Reagents and Solutions 9.10.10

Commentary 9.10.11

Transient Transfection Methods for Preparation of High-Titer
Retroviral Supematants 9.11.1

Basic Protocol I: Transient Transfection of a Retrovims Vector into
293 Cells 9.11.2

Support Protocol: Growth and Storage of 293 Cells 9.11.4

Basic Protocol 2: Pseudotyping a Stable Cell Line Sequentially with
VSV G Protein 9.11.6

Alternate Protocol I: Pseudotyping Cotransfectionally with VSV G Protein 9.11.8

Basic Protocol 3: Infection of Adherent Cells with Retroviral Supernatant 9.11.8

Alternate Protocol 2: Infection of Adherent Cells by Spin Infection 9.11.9

Basic Protocol 4: Infection of Nonadherent Cells by Retroviral Supernatant 9.11.10

Alternate Protocol 3: Infection of Nonadherent Cells by Cocultivation 9.11.11

Alternate Protocol 4: Infection of Nonadherent Cells by Spin Infection 9.11.12

Reagents and Solutions 9.11.13

Commentary 9.11.14


Large-Scale Preparation and Concentration of Retrovirus Stocks 9.12.1

Basic Protocol: Preparation of Virus Stock and Concentration
byCentrifugation 9.12.1

Alternate Protocol 1: Concentration by PEG Precipitation and
Chromatography 9.12.3

Alternate Protocol 2: Concentration Using Molecular-Weight-Cutoff Filters 9.12.5

Reagents and Solutions 9.12.5

Commentary 9.12.5

Detection of Helper Virus in Retrovirus Stocks 9.13.1

Basic Protocol: Detection of Helper Virus Through Horizontal Spread
of Drug Resistance 9.13.1

Alternate Protocol I: Proviral Rescue to Detect Replication-Competent
Retrovirus 9.13.3

Alternate Protocol 2: Reverse Transcriptase Assay to Detect Helper Virus 9.13.5

Reagents and Solutions 9.13.6

Commentary 9.13.6


Retrovirus Infection of Cells In Vitro and In Vivo 9.14.1

Infection of Cells In Vitro 9.14.1

Infection of Rodents and Chicks In Vivo 9.14.3

Literature Cited 9.14.5

GENE TARGETING BY HOMOLOGOUS RECOMBINATION

Overview of Gene Targeting by Homologous Recombination 9.15.1

Anatomy of Targeting Constructs 9.15.1

Methods of Enrichment for Homologous Recombinants 9.15.1

Types of Mutations 9.15.3

Production of a Heterozygous Mutant Cell Line by Homologous
Recombination (Single Knockout) 9.16.1

Strategic Planning 9.16.2

Basic Protocol: Gene Targeting in Embryonic Stem Cells ' 9.16.3

Reagents and Solutions 9.16.6

Commentary 9.16.7

Production of a Homozygous Mutant Embryonic Stem Cell
Line (Double Knockout) 9.17.1

Basic Protocol: Selection for Homozygous Clones 9.17.1

Commentary 9.17.2
ANALYSIS OF PROTEINS

INTRODUCTION 10.0.1

QUANTITATION OF PROTEINS

Spectrophotometric and Colorimetric Determination of Protein
Concentration 10.1.2

Basic Protocol I: Using A280 to Determine Protein Concentration 10.1.2

Alternate Protocol I: Using A 205 to Determine Protein Concentration 10.1.3

Basic Protocol 2: Using Fluorescence Emission to Determine Protein
Concentration 10.1.4

Basic Protocol 3: Using the Bradford Method to Determine Protein
Concentration 10.1.4

Alternate Protocol 2: Using the Lowry Method to Determine Protein
Concentration 10.1.5

Reagents and Solutions 10.1.6

Commentary 10.1.6

Quantitative Amino Acid Analysis 10.1.11

Information Obtained from Amino Acid Analysis 10.1.11

Sample Preparation 10.1.12

Calculation of the Average Composition 10.1.12

ELECTROPHORETIC SEPARATION OF PROTEINS

One-Dimensional Gel Electrophoresis of Proteins 10.2.2

Electricity and Electrophoresis 10.2.2

Basic Protocol I: Denaturing (SDS) Discontinuous Gel Electrophoresis:
Laemmii Gel Method 10.2.4

Alternate Protocol I: Electrophoresis in Tris-Tricine Buffer Systems 10.2.10

Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffer 10.2.12

Alternate Protocol 3: Continuous SDS-PAGE 10.2.14

Alternate Protocol 4: Casting and Running Ultrathin Gels 10.2.15

Support Protocol I: Casting Multiple Single-Concentration Gels 10.2.16

Alternate Protocol 5: Separations of Proteins on Gradient Gels 10.2.18

Support Protocol 2: Casting Multiple Gradient Gels 10.2.22

Basic Protocol 2: Electrophoresis in Single-Concentration Minigels 10.2.24

Support Protocol 3: Preparing Multiple Gradient Minigels 10.2.27

Reagents and Solutions 10.2.28

Commentary 10.2.30

Two-Dimensional Gel Electrophoresis Using the ISO-DALT System 10.3.1

Basic Protocol: First-Dimension (Isoelectric Focusing) Gels 10.3.1

Basic Protocol: Second-Dimension (Gradient) Gels 10.3.3

Alternate Protocols for Basic and Very Acidic Proteins 10.3.7

Support Protocol: Sample Solubilization 10.3.8

Short Protocol: Gel Electrophoresis Using the ISO-DALT System 10.3.9

Reagents and Solutions 10.3.10

Commentary 10.3.11

Two-Dimensional Gel Electrophoresis Using the CTFarrell System 10.4.1

Basic Protocol: First-Dimension (Isoelectric Focusing) Gels 10.4.1

Basic Protocol: Second-Dimension Gels 10.4.5

Alternate Protocols for First-Dimensional Gels: Isoelectric Focusing of
Very Basic and Very Acidic Proteins 10.4.8

NEPHGE for Very Basic Proteins 10.4.8

NEPHGE for Very Acidic Proteins 10.4.8

Alternate Protocol: Two-Dimensional Minigels 10.4.9

Support Protocol: Solubilization and Preparation of Proteins in
Tissue Samples 10.4.9

Reagents and Solutions 10.4.10

Commentary 10.4.11

DETECTION OF PROTEINS

Overview of Digital Electrophoresis Analysis 10.5.1

Reasons for Digital Documentation and Analysis 10.5.1

Key Terms for Imaging 10.5.2

Image Capture 10.5.3

Analysis 10.5.4


Staining Proteins in Gels 10.6.1

Basic Protocol: Coomassie Blue Staining 10.6.1

Alternate Protocol: Rapid Coomassie Blue Staining 10.6.2

Basic Protocol: Silver Staining 10.6.3

Alternate Protocol: Nonammoniacal Silver Staining 10.6.4

Alternate Protocol: Rapid Silver Staining 10.6.5

Support Protocol: Gel Photography 10.6.5

Reagents and Solutions 10.6.6

Commentary 10.6.7

Detection of Proteins on Blot Transfer Membranes 10.7.1

Basic Protocol: India Ink Staining 10.7.1

Alternate Protocol: Gold Staining 10.7.2

Support Protocol: Alkalai Enhancement of Protein Staining 10.7.2

Reagents and Solutions 10.7.2

Commentary 10.7.3

Immunoblotting and Immunodetection 10.8.1

Basic Protocol 1: Protein Blotting with Tank Transfer Systems 10.8.1

Alternate Protocol 1: Protein Blotting with Semidry Systems 10.8.4

Alternate Protocol 2: Blotting of Stained Gels 10.8.7

Support Protocol 1: Reversible Staining of Transferred Proteins 10.8.7

Support Protocol 2: Quantitation of Protein with Ponceau S 10.8.8

Basic Protocol 2: Immunoprobing with Directly Conjugated
Secondary Antibody 10.8.8

Alternate Protocol 3: Immunoprobing with Avidin-Biotin Coupling to
Secondary Antibody 10.8.10

Basic Protocol 3: Visualization with Chromogenic Substrates 10.8.12

Alternate Protocol 4: Visualization with Luminescent Substrates 10.8.13

Support Protocol 3: Stripping and Reusing Membranes 10.8.14

Reagents and Solutions 10.8.15

Commentary 10.8.16

PURIFICATION OF PROTEINS BY CONVENTIONAL
CHROMATOGRAPHY

Gel-Filtration Chromatography 10.9.3

Strategic Planning 10.9.3

Basic Protocol 1: Desalting (Group Separation) 10.9.8

Basic Protocol 2: Protein Fractionation 10.9.16

Basic Protocol 3: Determination of Molecular Size 10.9.19

Support Protocol: Column Calibration 10.9.21

Reagents and Solutions 10.9.24

Commentary 10.9.25


Ion-Exchange Chromatography 10.10.1

Strategic Planning 10.10.2

Basic Protocol 1: Batch Adsorption and Step-Gradient Elution with
Increasing Salt Concentration 10.10.10

Alternate Protocol: pH-Based Step-Gradient Elution 10.10.13

Basic Protocol 2: Column Chromatography with Linear Gradient Elution 10.10.13

Support Protocol I: Test Tube Pilot Experiment to Determine Starting
Conditions for I on-Exchange Chromatography 10.10.16

Support Protocol 2: Measurement of Dynamic (Column) Capacity and
Breakthrough Capacity of Ion-Exchange Columns 10.10.20

Support Protocol 3: Gradient-Formation Techniques 10.10.21

Support Protocol 4: Cleaning and Regeneration of lon-Exchange Media 10.10.23

Support Protocol 5: Storage of lon-Exchange Media 10.10.24

Commentary 10.10.25

Immunoaffinity Chromatography 10.11.1

Basic Protocol: Isolation of Soluble or Membrane-Bound Antigens 10.11.1

Alternate Protocol I: Low-pH Elution of Antigens 10.11.4

Alternate Protocol 2: Batch Purification of Antigens 10.11.5

Alternate Protocol 3: Elution in Octyl P-D-Glucoside 10.11.5

Reagents and Solutions 10.11.6

Commentary 10.11.7


Metal-Chelate Affinity Chromatography 10.11.10

Strategic Planning 10.11.10

Basic Protocol: Native MCAC for Purification of Soluble
Histidine-Tail Fusion Proteins 10.11.11

Alternate Protocol I: Denaturing MCAC for Purification of Insoluble
Histidine-Tail Fusion Proteins 10.11.15

Alternate Protocol 2: Solid-Phase Renaturation of MCAC-Purified
Proteins 10.11.17

Support Protocol 1: Analysis and Processing of Purified Proteins 10.11.18

Support Protocol 2: NTA Resin Regeneration 10.11.18

Reagents and Solutions 10.11.19

Commentary 10.11.21

PURIFICATION OF PROTEINS BY HIGH-PERFORMANCE
LIQUID CHROMATOGRAPHY

Reversed-Phase Isolation of Peptides 10.12.1

Basic Protocol 1: Reversed-Phase Peptide Separation at the
5-to500-pmolLevel 10.12.2

Basic Protocol 2: Reversed-Phase Peptide Separation at <5 pmol 10.12.4

Support Protocol: Capillary HPLC System Assembly 10.12.5

Basic Protocol 3: Peptide Mapping by Matrix-Assisted Laser
Desorption/lonization (MALDI) Mass Spectrometry 10.12.7

Alternate Protocol: Peptide Mapping by MALDI Mass Spectrometry
Using the Matrix Fast-Evaporation Method 10.12.9

Basic Protocol 4: Capillary Electrophoresis Analysis 10.12.10

Reagents and Solutions 10.12.11

Commentary 10.12.12

Ion-Exchange High-Performance Liquid Chromatography 10.13.1

Basic Protocol: Anion-Exchange HPLC 10.13.1

Basic Protocol: Cation-Exchange HPLC 10.13.3

Reagents and Solutions 10.13.3

Commentary 10.13.4

Size-Exclusion High-Performance Liquid Chromatography 10.14.1

Basic Protocol 10.14.1

Reagents and Solutions 10.14.3

Commentary 10.14.3

SPECIALIZED APPLICATIONS

Purification of Recombinant Proteins and Study of Protein
Interaction by Epitope Tagging 10.15.1

Basic Protocol 1: Immunoprecipitation ofEpitope-Tagged Recombinant
Proteins 10.15.2

Basic Protocol 2: Immunoprecipitation ofEpitope-Tagged Recombinant
Proteins from a Baculovirus Overexpression System 10.15.4

Alternate Protocol: Stepwise Assembly of Protein Complexes 10.15.5

Reagents and Solutions 10.15.6

Commentary 10.15.7

Immunoprecipitation 10.16.1

Basic Protocol: Immunoprecipitation of Radiolabeled Antigen with
Antibody-Sepharose 10.16.1

Support Protocol: Preparation of Antibody-Sepharose 10.16.4

Alternate Protocol: Immunoprecipitation of Radiolabeled Antigen
with Anti-lg Serum 10.16.5

Alternate Protocol: Immunoprecipitation of Radiolabeled Antigen

with Anti-lg-Sepharose, Protein A- or G-Sepharose,
or S. Aureus Cells 10.16.6

Alternate Protocol: Immunoprecipitation Using More Strongly
Dissociating Lysis and Wash Buffers 10.16.7

Alternate Protocol: Immunoprecipitation of Unlabeled Antigen
with Antibody-Sepharose 10.16.7

Reagents and Solutions 10.16.8

Commentary 10.16.9
Synthesizing Proteins In Vitro by Transcription and
TYanslation of Cloned Genes 10.17.1

Basic Protocol 10.17.1

Reagents and Solutions 10.17.3

Commentary 10.17.3

Metabolic Labeling with Amino Acids 10.18.1

Safety Precautions for Working with S-Labeled Compounds 10.18.2

Basic Protocol: Pulse-Labeling of Cells in Suspension with [35 S]Methionine 10.18.2

Alternate Protocol I: Pulse-Labeling of Adherent Cells with [35 S]Methionine 10.18.4

Alternate Protocol 2: Pulse-Chase Labeling of Cells with [^S]Methionine 10.18.5

Alternate Protocol 3: Long-Term Labeling of Cells with [^S]Methionine 10.18.5

Alternate Protocol 4: Metabolic Labeling with Other Radiolabeled Amino Acids 10.18.6

Support Protocol: TCA Precipitation to Determine Label Incorporation 10.18.7

Reagents and Solutions 10.18.7

Commentary 10.18.8


Isolation of Proteins for Microsequence Analysis 10.19.1

Basic Protocol: Determination of Amino Acid Sequence by SDS-PAGE
and Transfer to PVDF Membranes 10.19.1

Support Protocol: Preparation of Protein Samples for SDS-PAGE 10.19.4

Basic Protocol: Determination of Internal Amino Acid Sequence from
Electrophoretically Separated Proteins 10.19.5

Reagents and Solutions 10.19.8

Commentary 10.19.9

Capillary Electrophoresis of Proteins and Peptides 10.20.1

Instrumentation 10.20.2

Separation Theory 10.20.3

Strategic Planning 10.20.4

Basic Protocol 1: Separation of Protein by Isoelectric Focusing 10.20.5

Basic Protocol 2: Separation of Proteins 10.20.6

Basic Protocol 3: Analytical Peptide Separations 10.20.8

Basic Protocol 4: Micropreparative Capillary Electrophoresis:
Multiple Separations 10.20.9

Alternate Protocol: Micropreparative Capillary Electrophoresis:
Single Separation 10.20.10

Commentary 10.20.11

Overview of Peptide and Protein Analysis by Mass Spectrometry 10.21.1

Why is MS an Essential Tool in Protein Structure Analysis? 10.21.1

What is MS? 10.21.3

Key lonization Methods and Related Ancillary Techniques 10.21.3

What is Tandem MS? 10.21.4

Is MS Data Quantitative? 10.21.15

Sample Preparation 10.21.15

Fundamentals of Mass Measurement Accuracy and Mass Resolution 10.21.17

Outlook 10.21.22

Protein Identification and Characterization by Mass Spectrometry 10.22.1

Protein Identification Using MS-Based Correlative Approaches 10.22.2

Direct MS Analysis of Gel-Separated Proteins and Other Multi-Dimensional
Approaches for Protein Separation and Mass Measurement 10.22.9

Rapid Posttranslational Modification Analysis , 10.22.13

High-Throughput Protein Identification 10.22.17

Conclusion 10.22.21

IMMUNOLOGY

INTRODUCTION 11.0.3
IMMUNOASSAYS

Conjugation of Enzymes to Antibodies 11.1.1

Basic Protocol: Conjugation of Horseradish Peroxidase to Antibodies 11.1.2

Alternate Protocol: Conjugation of Urease to Antibodies 11.1.3

Alternate Protocol: Conjugation of Alkaline Phosphatase to Antibodies 11.1.4

Reagents and Solutions 11.1.5

Commentary 11.1.6

Enzyme-Linked Immunosorbent Assay (ELISA) 11.2.1

Basic Protocol: Indirect ELISA to Detect Specific Antibodies 11.2.2

Alternate Protocol: Direct Competitive ELISA to Detect Soluble Antigens 11.2.5

Alternate Protocol: Antibody-Sandwich ELISA to Detect Soluble Antigens 11.2.8

Alternate Protocol: Double Antibody-Sandwich ELISA to Detect
Specific Antibodies 11.2.10

Alternate Protocol: Direct Cellular ELISA to Detect Cell-Surface Antigens 11.2.12

Alternate Protocol: Indirect Cellular ELISA to Detect Antibodies
Specific for Surface Antigens 11.2.14

Support Protocol: Criss-Cross Serial-Dilution Analysis to Determine
Optimal Reagent Concentrations 11.2.16

Support Protocol: Preparation of Bacterial Cell Lysate Antigens 11.2.18

Reagents and Solutions 11.2.18

Commentary 11.2.19

Isotype Determination of Antibodies 11.3.1

Basic Protocol: Sandwich ELISA for Isotype Detection 11.3.1

Basic Protocol: Detecting and Isotyping Antibodies by Electrophoresis
and Immunofixation 11.3.2

Reagents and Solutions 11.3.4

Commentary 11.3.4


PREPARATION OF MONOCLONAL ANTIBODIES

Immunization of Mice 11.4.2

Basic Protocol: Production of Immune Spleen Cells: Immunization with
Soluble Antigen 11.4.2

Alternate Protocol: Immunization with Complex Antigens (Membranes,
Whole Cells, and Microorganisms) 11.4.4

Alternate Protocol: Immunization with Antigen Isolated by Electrophoresis 11.4.4

Commentary 11.4.5

Preparation of Myeloma Cells 11.5.1

Basic Protocol 11.5.1

Support Protocol: Cell Viability Test by Trypan Blue Exclusion 11.5.1

Reagents and Solutions 11.5.2

Commentary 11.5.3


11.6 Preparation of Mouse Feeder Cells for Fusion and Cloning 11.6.1

Basic Protocol 11.6.1

Reagents and Solutions 11.6.2

Commentary , 11.6.3


Fusion of Myeloma Cells with Immune Spleen Cells 11.7.1

Basic Protocol 11.7.1

Reagents and Solutions 11.7.3

Commentary 11.7.4


Cloning of Hybridoma Cell Lines by Limiting Dilution 11.8.1

Basic Protocol 11.8.1

Commentary 11.8.2

Freezing and Recovery of Hybridoma Cell Lines 11.9.1

Basic Protocol: Freezing of Cell Lines 11.9.1

Basic Protocol: Recovery of Frozen Cell Lines 11.9.1

Reagents and Solutions 11.9.2

Commentary 11.9.3

Production of Monoclonal Antibody Supernatant and Ascites Fluids 11.10.1

Basic Protocol: Production of a Monoclonal Antibody Supernatant 11.10.1

Alternate Protocol: Large-Scale Production of Monoclonal Antibody
Supernatant 11.10.2

Alternate Protocol: Large-Scale Production of Hybridornas or Cell Lines 11.10.3

Basic Protocol: Production of Ascites Fluid Containing Monoclonal
Antibody 11.10.4

Commentary 11.10.6

Purification of Monoclonal Antibodies 11.11.1

Basic Protocol: Purification Using Protein A-Sepharose 11.11.1

Alternate Protocol: Alternative Buffer System for Protein A-Sepharose 11.11.2

Alternate Protocol: Purification by Antigen-Sepharose and Anti-mouse
Immunoglobulin-Sepharose 11.11.2

Reagents and Solutions 11.11.4

Commentary 11.11.4


PREPARATION OF POLYCLONAL ANTISERA

Production of Polyclonal Antiscra 11.12.1

Strategic Planning 11.12.1

Basic Protocol: Immunization to Produce Polyclonal Antibodies
Using Freund's Adjuvant 11.12.2

Alternate Protocol: Immunization to Produce Polyclonal Antiserum
Using Other Adjuvants 11.12.4

Support Protocol: Preparation of Serum from Blood 11.12.5

Commentary 11.12.6


Purification of Immunoglobulin G Fraction from Antiserum,
Ascites Fluid, or Hybridoma Supernatant 11.13.1

Basic Protocol: Precipitation ofIgG with Saturated Ammonium Sulfate 11.13.1

Alternate Protocol: Fractionation ofIgG by Chromatography
on DEAE-Affi-Gel Blue 11.13.1

Reagents and Solutions 11.13.2

Commentary 11.13.3


PREPARATION OF ANTIPEPTIDE ANTIBODIES
Selection of an Inimunogenic Peptide 11.14.1

Production of Antipeptide Antibodies 11.15.1

Basic Protocol: Chemical Coupling of Synthetic Peptide to Carrier
Protein Using MBS 11.15.1

Alternate Protocol: Chemical Coupling of Synthetic Peptide to Carrier
Protein Using Glutaraldehyde 11.15.2

Reagents and Solutions 11.15.3

Commentary 11.15.3

DETERMINATION OF SPECIFIC ANTIBODY TITER AND ISOTYPE

Determination of the SpeciTic Antibody Titer 11.16.1

Basic Protocol: Solid-Phase Radioimmunoassay (RIA) for Determination
of Antibody Titer 11.16.1

Support Protocol: lodination ofAnti-lmmunoglobulin Antibodies Using
ChloramineT 11.16.5

Support Protocol: lodination ofAnti-lmmunoglobulin Antibodies
Using IODO-GEN 11.16.7

Support Protocol: Determination of Antibody Isotypes 11.16.7

Reagents and Solutions 11.16.10

Commentary 11.16.11

DNA-PROTEIN
INTERACTIONS

INTRODUCTION 12.0.3

Preparation of Nuclear and Cytoplasmic Extracts from
Mammalian Cells 12.1.1

Basic Protocol: Preparation of Nuclear Extracts 12.1.1

Support Protocol: Optimization of Nuclear Extraction 12.1.4

Support Protocol: Preparation of the Cytoplasmic (S-100) Fraction 12.1.4

Reagents and Solutions 12.1.5

Commentary 12.1.6

Mobility Shift DNA-Binding Assay Using Gel Electrophoresis 12.2.1

Strategic Planning 12.2.1

Basic Protocol: Mobility Shift Assay 12.2.2

Alternate Protocol 1: Competition Mobility Shift Assay 12.2.5

Alternate Protocol 2: Antibody Supershift Assay 12.2.5

Alternate Protocol 3: Multicomponent Mobility Shift Assaya 12.2.6

Reagents and Solutions 12.2.7

Commentary 12.2.7

Methylation and Uracil Interference Assays for Analysis of
Protein-DNA Interactions 12.3.1

Basic Protocol: Methylation Interference Assay 12.3.1

Basic Protocol: Uracil Interference Assay 12.3.3

Reagents and Solutions 12.3.5

Commentary 12.3.5


DNase I Footprint Analysis of Protein-DNA Binding 12.4.1

Basic Protocol: DNase I Footprint Titration 12.4.1

Support Protocol: Quantitation of Protein-Binding Equilibria by
Densitometric and Numerical Analyses 12.4.6

Alternate Protocol: DNase Footprinting in Crude Fractions 12.4.9

Reagents and Solutions 12.4.11

Commentary 12.4.11

UV Crosslinking of Proteins to Nucicic Acids 12.5.1

Basic Protocol: UV Crosslinking Using a Bromodeoxyuridine-
Substituted Probe 12.5.1

Alternate Protocol: UV Crosslinking Using a Non-Bromodeoxyuridine-
Substituted Probe 12.5.4

Alternate Protocol: UV Crosslinking in Situ 12.5.4

Reagents and Solutions 12.5.5

Commentary 12.5.6


Purification of DNA-Binding Proteins Using
Biotin/Streptavidin Affinity Systems 12.6.1

Basic Protocol 12.6.1

Alternate Protocol: Purification Using a Microcolumn 12.6.4

Alternate Protocol: Purification Using Streptavidin-Agarose 12.6.5

Reagents and Solutions , 12.6.6

Commentary 12.6.6

Detection, Purification, and Characterization of cDNA Clones
Encoding DNA-Binding Proteins 12.7.1

Basic Protocol: Screening a (lambdal) gtil Expression Library with
Recognition-Site DNA 12.7.1

Alternate Protocol: Denaturation / Renaturation Cycling of Dried Replica
Filters Using Guanidine'HCI 12.7.4

Support Protocol: Preparation of a Crude Extract from a (lambdal) gtll
Recombinant Lysogen to Characterize DNA-Binding Activity
of the Fusion Protein 12.7.5

Reagents and Solutions 12.7.6

Commentary 12.7.7

Rapid Separation of Protein-Bound DNA from Free DNA
Using Nitrocellulose Filters 12.8.1

Basic Protocol 12.8.1

Alternate Protocol: Detection of Specificity in DNA Binding 12.8.2

Support Protocol: Elution of Bound DNA 12.8.4

Reagents and Solutions 12.8.5

Commentary 12.8.5

Analysis of DNA-Protein Interactions Using Proteins
Synthesized In Vitro from Cloned Genes 12.9.1

Basic Protocol 12.9.1

Reagents and Solutions 12.9.2

Commentary 12.9.2

Purification of Sequence-Specific DNA-Binding Proteins
by Affinity Chromatography 12.10.1

Basic Protocol: Preparation of DNA Affinity Resin 12.10.1

Alternate Protocol: Coupling the DNA to Commercially Available
CNBr-Activated Sepharose 12.10.6

Support Protocol: Purification ofOligonucleotides by Preparative
Gel Electrophoresis 12.10.7

Basic Protocol: DNA Affinity Chromatography 12.10.9

Support Protocol: Selection and Preparation of Nonspecific
Competitor DNA 12.10.11

Reagents and Solutions 12.10.12

Commentary 12.10.14

Determination of Protein-DNA Sequence Specificity by
PCR-Assisted Binding-Site Selection 12.11.1

Basic Protocol 12.11.1

Support Protocol: Isolation and Analysis of Bound Oligonucleotides
from Mobility Shift Gels 12.11.6

Reagents and Solutions 12.11.7

Commentary 12.11.8
DNA-PROTEIN
INTERACTIONS

INTRODUCTION 12.0.3

Preparation of Nuclear and Cytoplasmic Extracts from
Mammalian Cells 12.1.1

Basic Protocol: Preparation of Nuclear Extracts 12.1.1

Support Protocol: Optimization of Nuclear Extraction 12.1.4

Support Protocol: Preparation of the Cytoplasmic (S-100) Fraction 12.1.4

Reagents and Solutions 12.1.5

Commentary 12.1.6

Mobility Shift DNA-Binding Assay Using Gel Electrophorcsis 12.2.1

Strategic Planning 12.2.1

Basic Protocol: Mobility Shift Assay 12.2.2

Alternate Protocol 1: Competition Mobility Shift Assay 12.2.5

Alternate Protocol 2: Antibody Supershift Assay 12.2.5

Alternate Protocol 3: Multicomponent Mobility Shift Assay a 12.2.6

Reagents and Solutions 12.2.7

Commentary 12.2.7

Methylation and Uracil Interference Assays for Analysis of
Protein-DNA Interactions 12.3.1

Basic Protocol: Methylation Interference Assay 12.3.1

Basic Protocol: Uracil Interference Assay 12.3.3

Reagents and Solutions 12.3.5

Commentary 12.3.5


DNase I Footprint Analysis of Protein-DNA Binding 12.4.1

Basic Protocol: DNase I Footprint Titration 12.4.1

Support Protocol: Quantitation of Protein-Binding Equilibria by
Densitometric and Numerical Analyses 12.4.6

Alternate Protocol: DNase Footprinting in Crude Fractions 12.4.9

Reagents and Solutions 12.4.11

Commentary 12.4.11

UV Crosslinking of Proteins to Nucleic Acids 12.5.1

Basic Protocol: UV Crosslinking Using a Bromodeoxyuridine-
Substituted Probe 12.5.1

Alternate Protocol: UV Crosslinking Using a Non-Bromodeoxyuridine-
Substituted Probe 12.5.4

Alternate Protocol: UV Crosslinking in Situ 12.5.4

Reagents and Solutions 12.5.5

Commentary 12.5.6

Purification of DNA-Binding Proteins Using

Biotin/Streptavidin Affinity Systems 12.6.1

Basic Protocol 12.6.1

Alternate Protocol: PuriFication Using a Microcolumn , 12.6.4

Alternate Protocol: Purification Using Streptavidin-Agarose 12.6.5

Reagents and Solutions 12.6.6

Commentary 12.6.6

SACCHARÖMYCES CEREVISIAE

INTRODUCTION 13.0.1
BASIC TECHNIQUES OF YEAST GENETICS

Preparation of Yeast Media 13.1.1

Liquid Media 13.1.2

Solid Media 13.1.4

Strain Storage and Revival 13.1.6

Basic Protocol: Preparation and Inoculation of Frozen Stocks 13.1.6

Alternate Protocol: Preparation and Inoculation of Slants 13.1.6

Basic Protocol: Mailing and Reviving Strains 13.1.7

Growth and Manipulation of Yeast 13.2.1

Basic Protocol: Growth in Liquid Media 13.2.1

Basic Protocol: Growth on Solid Media 13.2.1

Basic Protocol: Determination of Cell Density 13.2.1

Basic Protocol: Determination of Phenotype by Replica Plating 13.2.2

Basic Protocol: Determination of Mating Type 13.2.2

Strain Construction and Tetrad Analysis 13.2.3

Basic Protocol: Diploid Construction 13.2.4

Basic Protocol: Sporulation on Plates and in Liquid Media 13.2.4

Basic Protocol: Preparation and Dissection of Tetrads 13.2.5

Support Protocol: Preparation of Dissecting Needles 13.2.8

Alternate Protocol: Random Spore Analysis 13.2.9

Commentary 13.2.10

Mutagencsis of Yeast Cells 13.3.1

Basic Protocol: Mutagenesis Using Ethyl Methanesulfonate (EMS) 13.3.1

Alternate Protocol: Mutagenesis Using UV Irradiation 13.3.3

Reagents and Solutions 13.3.3

Commentary 13.3.4


YEAST VECTORS

Yeast Cloning Vectors and Genes 13.4.1

Plasmid Nomenclature 13.4.2

Maps of Selected Plasmids and Genes 13.4.3


Yeast Vectors for Expression of Cloned Genes
To come in later supplement

Yeast Vectors and Assays for Expression of Cloned Genes 13.6.1

Basic Protocol 1: Construction of lacz Fusion Vectors for Studying Yeast
Gene Regulation 13.6.2

Basic Protocol 2: Assay for P-galactosidase in Liquid Cultures 13.6.2

Alternate Protocol: Screening for P-galactosidase-Expressing Yeast Colonies
Using a Filter Lift Assay 13.6.4

Reagents and Solutions 13.6.5

Commentary 13.6.5

MANIPULATION OF YEAST GENES

Introduction of DNA into Yeast Cells 13.7.1

Basic Protocol: Transformation Using Lithium Acetate 13.7.1

Alternate Protocol: Spheroplast Transformation 13.7.3

Alternate Protocol: Transformation by Electroporation 13.7.5

Support Protocol: Preparation of Single-Stranded High-Molecular
Weight Carrier DNA 13.7.7

Reagents and Solutions 13.7.8

Commentary 13.7.9


Cloning Yeast Genes by Complementation 13.8.1

Basic Protocol 13.8.1

Commentary 13.8.3


Manipulation of Plasmids from Yeast Cells 13.9.1

Basic Protocol I: Segregation of Plasmids from Yeast Cells 13.9.1

Basic Protocol 2: Plasmid Shuffling 13.9.2

Basic Protocol 3: Plasmid Gap Repair for Localized Mutagenesis and
Allele Repair 13.9.4

Commentary 13.9.5


Manipulation of Cloned Yeast DNA 13.10.1

Basic Protocol I: Integrative Transformation 13.10.1

Gene Replacement Techniques 13.10.2

Basic Protocol 2: Integrative Disruption 13.10.3

Basic Protocol 3: One-Step Gene Disruption 13.10.4

Alternate Protocol I: PCR-Mediated One-Step Gene Disruption 13.10.5

Basic Protocol 4: Transplacement 13.10.8

Basic Protocol 5: Creating Modified Genes by One-Step Integrative
Replacement 13.10.9

Alternate Protocol 2: Creating Modified Genes by Transplacement 13.10.11

Basic Protocol 6: Creation of Conditional Alleles by Copper-inducible
Double-Shutoff Procedure 13.10.11

PREPARATION OF YEAST DNA, RNA, AND PROTEINS

Preparation of Yeast DNA 13.11.1

Basic Protocol: Rapid Isolation of Plasmid DNA from Yeast 13.11.1

Alternate Protocol: Rapid Isolation of Yeast Chromosomal DNA 13.11.2

Reagents and Solutions 13.11.3

Commentary 13.11.3


Preparation of Yeast RNA 13.12.1

Basic Protocol: Preparation of Yeast RNA by Extraction with Hot
Acidic Phenol 13.12.1

Alternate Protocol: Preparation of RNA Using Glass Beads 13.12.2

Alternate Protocol: Preparation of Poly(A)+ RNA 13.12.4

Reagents and Solutions 13.12.4

Commentary 13.12.4


Preparation of Protein Extracts from Yeast 13.13.1

Basic Protocol: Spheroplast Preparation and Lysis 13.13.1

Support Protocol: Nuclei Preparation by Differential Centrifugation 13.13.3

Alternate Protocol: Cell Disruption Using Glass Beads 13.13.4

Alternate Protocol: Cell Disruption Using Liquid Nitrogen 13.13.5

Reagents and Solutions 13.13.6

Commentary 13.13.7

IN SITU HYBRIDIZATION AND
IMMÜNOHISTOCHEMISTRY

INTRODUCTION 14.0.1

Fixation, Embedding, and Sectioning of Tissues, Embryos,
and Single Cells 14.1.1

Basic Protocol: Paraformaldehyde Fixation and Paraffin Wax Embedding
of Tissues and Embryos 14.1.1

Alternate Protocol: Fixation of Suspended and Cultured Cells 14.1 .3

Support Protocol: Perfusion of Adult Mice 14.1 .4

Support Protocol: Sectioning Samples in Wax Blocks 14.1 .4

Reagents and Solutions 14.1 .6

Commentary 14.1 .7

Cryosectioning 14.2.1

Basic Protocol: Specimen Preparation and Sectioning 14.2.1

Support Protocol: Fixation of Cryosections for In situ Hybridization 14.2.5

Support Protocol: Tissue Fixation and Sucrose Infusion 14.2.6

Commentary 14.2.6

In situ Hybridization to Cellular RNA 14.3.1

Basic Protocol: Hybridization Using Paraffin Sections and Cells 14.3.1

Alternate Protocol: Hybridization Using Cryosections 14.3.5

Support Protocol: Synthesis of (35)S-Labeled Riboprobes 14.3.7

Support Protocol: Synthesis of (35)S-Labeled Double-Stranded DNA Probes 14.3.8

Reagents and Solutions 14.3.8

Commentary 14.3.11


Detection of Hybridized Probe 14.4.1

Basic Protocol: Film Autoradiography 14.4.1

Basic Protocol: Emulsion Autoradiography 14.4.1

Support Protocol: Preparation of Diluted Emulsion for Autoradiography 14.4.2

Commentary 14.4.3


Counterstaining and Mounting of Autoradiographed
In situ Hybridization Slides 14.5.1


Basic Protocol: Giemsa Staining 14.5.1

Alternate Protocol: Hematoxylin/Eosin Staining 14.5.2

Alternate Protocol: Toluidine Blue Staining 14.5.3

Alternate Protocol: Hoechst Staining 14.5.3

Reagents and Solutions 14.5.4

Commentary 14.5.5


Immunohistochemistry 14.6.1


Basic Protocol: Immunofluorescent Labeling of Cells Grown as
Monolayers 14.6.1

Alternate Protocol: Immunofluorescent Labeling of Suspension Cells 14.6.2

Basic Protocol: Immunofluorescent Labeling of Tissue Sections 14.6.3

Alternate Protocol: Immunofluorescent Labeling of Tissue Sections
Using Coplin Jars 14.6.4

Alternate Protocol: Immunofluorescent Labeling Using
Streptavidin-Biotin Conjugates 14.6.5

Alternate Protocol: Immunogold Labeling of Tissue Sections 14.6.6

Alternate Protocol: Immunoperoxidase Labeling of Tissue Sections 14.6.6

Alternate Protocol: Immunofluorescent Double-Labeling of Tissue Sections 14.6.7

Reagents and Solutions 14.6.7

Commentary 14.6.8
In situ Hybridization and Detection Using Nonisotopic Probes 14.7.1

Basic Protocol 1: Fluorescence in situ Hybridization 14.7.1

Amplification of Hybridization Signals 14.7.4

Support Protocol I: Amplification of Biotinylated Signals 14.7.4

Support Protocol 2: Amplification of Signals from Digoxigenin-Labeled
Probes 14.7.5

Enzymatic Detection of Nonisotopically Labeled Probes 14.7.6

Alternate Protocol 1: Enzymatic Detection Using Horseradish Peroxidase 14.7.7

Alternate Protocol 2: Enzymatic Detection Using Alkaline Phosphatase 14.7.8

Reagents and Solutions 14.7.9

Commentary 14.7.11

In situ Polymerase Chain Reaction and Hybridization to Detect
Low-Abundance Nucleic Targets 14.8.1

Strategic Planning 14.8.1

Basic Protocol 1: In situ PCR (ISPCR) Amplification ofDNA and RNA
Targets with in situ Reverse Transcriptional for RNA 14.8.6

Alternate Protocol: One-Step Reverse Transcription and Amplification 14.8.10

Basic Protocol 2: Hybridization and Detection ofISPCR-Amplified
Target Material 14.8.11

Support Protocol 1: Preparation of AES-Subbed Slides 14.8.14

Support Protocol 2: Preparation of Specimens on Slides for ISPCR 14.8.14

Support Protocol 3: Labeling Oligosaccharide Probes Using (33)P 14.8.16

Reagents and Solutions 14.8.16

Commentary 14.8.18

Basic Protocol 2: Enzymatic Detection of RNA Hybrids in Mouse and
Chicken Embryos and Organs 14.9.4

Alternate Protocol 1: Whole-Mount in situ Hybridization with XENÖPUS
Embryos 14.9.7

Alternate Protocol 2: Enzymatic Detection of RNA Hybrids in XENÖPUS
Embryos 14.9.9

Support Protocol 1: Synthesis of Digoxigenin-Labeled RNA Probes 14.9.10

Support Protocol 2: Preabsorption of Fab Fragments with Embryonic
Powder 14.9.11

Reagents and Solutions 14.9.13

Commentary 14.9.14


Principles and Application of Fluorescence Microscopy 14.10.1

Fluorescent Molecular Probes 1 4.10.2

Filters and Filter Sets 14.10.2

Multiband Filters and Multidye Fluorescence 14.10.3

Light Sources 14.10.4

Microscope Objectives 14.10.6

Image Resolution and the Point-Spread Function (PSF) 14.10.6

Fluorescence Microscopy of Living Cells 14.10.7

Immunolabeling: General Steps for Labeling Fixed Cells and Tissues 14.10.8


Basic Confocal Microscopy 14.11.1

The Basis of Optical Sectioning 14.11.1

Types of Confocal Microscopes 14.11.3

Practical Guidelines 14.11.6

THE POLYMERASE
CHAIN REACTION

INTRODUCTION 15.0.3

Enzymatic Amplification of DNA by PCR:

Standard Procedures and Optimization 15.1.1

Basic Protocol 15.1.1

Reagents and Solutions 15.1.5

Commentary 15.1.5

Direct DNA Sequencing of PCR Products 15.2.1

Basic Protocol: Generating Single-Stranded Products for
Dideoxy Sequencing by Asymmetric PCR 15.2.1

Alternate Protocol: Generating Single-Stranded Template for
Dideoxy Sequencing by Single-Primer Reamplification 15.2.3

Alternate Protocol: Preparing Double-Stranded PCR Products for
Dideoxy Sequencing 15.2.4

Alternate Protocol: Generating Single-Stranded Template for Dideoxy
Sequencing by(lambdal) Exonuclease Digestion of Double-Stranded
PCR Products 15.2.5

Basic Protocol: Labeling PCR Products for Chemical Sequencing 15.2.6

Alternate Protocol: Genomic Sequencing of PCR Products 15.2.7

Reagents and Solutions 15.2.9

Commentary 15.2.9

QuantitationofRareDNAsbyPCR 15.3.1

Basic Protocol 15.3.1

Reagents and Solutions 15.3.5

Commentary 15.3.5


Enzymatic Amplification of RNA by PCR 15.4.1

Basic Protocol: PCR Amplification of RNA Under Optimal Conditions 15.4.1

Alternate Protocol: Avoiding Lengthy Coprecipitation and Annealing Steps 15.4.3

Alternate Protocol: Introducing cDNA Directly into the Amplification Step 15.4.3

Support Protocol: Rapid Precipitation of Crude RNA 15.4.4

Reagents and Solutions 15.4.4

Commentary 15.4.5

Ligation-Mediated PCR for Genomic Sequencing and Footprinting 15.5.1

Basic Protocol: Ligation-Mediated Single-Sided PCR 15.5.1

Support Protocol: Preparation of Genomic DNA from Monolayer Cells
for DMS Footprinting 15.5.8

Support Protocol: Preparation of Genomic DNA from Suspension Cells
for DMS Footprinting 15.5.14

Support Protocol: Preparation of Genomic DNA for Chemical Sequencing 15.5.15

Reagents and Solutions 15.5.17

Commentary 15.5.20

cDNA Amplification Using Onc-Sided (Anchored) PCR 15.6.1

Basic Protocol: Amplification of Regions Downstream (3') of Known Sequence 15.6.1

Basic Protocol: Amplification of Regions Upstream (5') of Known Sequence 15.6.4

Reagents and Solutions 15.6.7

Commentary 15.6.8
Molecular Cloning of PCR Products 15.7.1

Basic Protocol: Generation ofT-A Overhangs 15.7.1

Alternate Protocol 1: Generation of Half-Sites 15.7.3

Alternate Protocol 2: Cloning PCR Products with Uracil DNA Glycosylase 15.7.5

Support Protocol: Designing Primer Sets for Amplification and Construction
of LJDG Cloning Vectors 15.7.7

Commentary 15.7.8

Differential Display of mRNA by PCR 15.8.1

Basic Protocol 15.8.1

Reagents and Solutions 15.8.7

Commentary 15.8.7
PROTEIN EXPRESSION

INTRODUCTION 16.0.5

EXPRESSION OF PROTEINS IN ESCHERICHIACOLI

Overview of Protein Expression in E. coli 16.1.1

General Strategy for Gene Expression in E. coli 16.1.1

Specific Expression Scenarios 16.1.1

Troubleshooting Gene Expression 16.1.2

Expression Using the T7 RNA Polymerase/Promoter System 16.2.1

Basic Protocol: Expression Using the Two-Plasmid System 16.2.1

Alternate Protocol: Selective Labeling of Plasmid-Encoded Proteins 16.2.5

Alternate Protocol: Expression by Infection with M13 Phage mGPI-2 16.2.6

Reagents and Solutions 16.2.8

Commentary 16.2.8

Expression Using Vectors with Phage(lambdal) Regulatory Sequences 16.3.1

Basic Protocol: Temperature Induction of Gene Expression 16.3.1

Basic Protocol: Chemical Induction of Gene Expression 16.3.2

Support Protocol: Authentic Gene Cloning Using pSKF Vectors 16.3.3

Support Protocol: Construction and Disassembly of Fused Genes
in pSKF301 16.3.6

Commentary 16.3.8

Introduction to Expression by Fusion Protein Vectors 16.4.1

Solubility of the Expressed Protein 16.4.1

Stability of the Expressed Protein 16.4.2

Cleavage of Fusion Proteins to Remove the Carrier 16.4.2

Enzymatic and Chemical Cleavage of Fusion Proteins 16.4.5

Basic Protocol I: Enzymatic Cleavage of Fusion Proteins with Factor Xa 16.4.6

Support Protocol: Denaturing a Fusion Protein for Factor Xa Cleavage 16.4.7

Alternate Protocol 1: Enzymatic Cleavage of Fusion Proteins
withThrombin 16.4.8

Alternate Protocol 2: Enzymatic Cleavage of Matrix-Bound GST
Fusion Proteins 16.4.9

Alternate Protocol 3: Enzymatic Cleavage of Fusion Proteins
withEnterokinase 16.4.10

Basic Protocol 2: Chemical Cleavage of Fusion Proteins Using
Cyanogen Bromide 16.4.11

Alternate Protocol 4: Chemical Cleavage of Fusion Proteins
Using Hydroxiamine 16.4.12

Alternate Protocol 5: Cleavage of Fusion Proteins by Hydrolysis
atLowpH 16.4.13

Reagents and Solutions 16.4.14

Commentary 16.4.14

Expression and Purification of lacZ and trpE Fusion Proteins 16.5.1


Basic Protocol 16.5.1

Reagents and Solutions 16.5.4

Commentary 16.5.4
Expression and Purification of Maltose-Binding Protein Fusions 16.6.1

Basic Protocol: Construction, Expression, and Purification of MBP
Fusion Proteins 16.6.3

Support Protocol I: Pilot Experiment to Characterize the Behavior of an
MBP Fusion Protein 16.6.5

Alternate Protocol: Purification of Fusion Proteins from the Periplasm 16.6.7

Support Protocol 2: Purifying the Cleaved Protein by Ion Exchange
Chrornatography 16.6.8

Support Protocol 3: Purifying the Cleaved Protein by Affinity
Chromatography 16.6.9

Reagents and Solutions 16.6.10

Commentary 16.6.10

Expression and Purification of Glutathione-S-Transferase
Fusion Proteins 16.7.1

Basic Protocol 16.7.1

Reagents and Solutions 16.7.5

Commentary 16.7.5


Expression and Purification of Thioredoxin Fusion Proteins 16.8.1

Strategic Planning 16.8.1

Basic Protocol: Construction and Expression of a Thioredoxin
Fusion Protein 16.8.4

Support Protocol 1: E. Coli Lysis Using a French Pressure Cell 16.8.6

Support Protocol 2: Osmotic Release of Thioredoxin Fusion Proteins 16.8.8

Support Protocol 3: Purification of Thioredoxin Fusion Proteins by
Heat Treatment 16.8.9

Reagents and Solutions 16.8.10

Commentary 16.8.11


EXPRESSION OF PROTEINS IN INSECT CELLS
USING BACULOVIRUS VECTORS

Overview of the Baculovirus Expression System 16.9.1

Baculovirus Life Cycle 16.9.1

Baculovirus Expression System 16.9.2

Post-Translational Modification of Proteins in Insect Cells 16.9.3

Steps for Overproducing Proteins Using the Baculovirus Expression System 16.9.4

Choosing a Baculovirus Transfer Vector 16.9.4

Choosing a Baculvirus DNA 16.9.8

Reagents, Solutions, and Equipment for the Baculovirus Expression System 16.9.9

Maintenance of Insect Cell Cultures and Generation of Recombinant
Baculoviruses 16.10.1

Basic Protocol 1: Maintenance and Culture of Insect Cells 16.10.1

Basic Protocol 2: Cotransfection of Insect Cells Using Linearized
Baculoviral DNA 16.10.3

Alternate Protocol 1: Generation of Recombinant Baculovirus Using
Wild-Type Baculoviral DNA 16.10.6

Alternate Protocol 2: Generation of Recombinant Baculoviruses by Direct
Cloning 16.10.9

Basic Protocol 3: Preparation of Baculovirus Stocks 16.10.10

Basic Protocol 4: Titering Baculovirus Stocks Using Plaque Assay 16.10.12

Reagents and Solutions 16.10.14

Commentary 16.10.15
Expression and Purification of Recombinant Proteins Using the
Baculovirus System 16.11.1

Basic Protocol 1: Small-Scale Expression for Initial Analysis 16.11.1

Support Protocol 1: Determining Time Course of Maximum Protein
Production 16.11.3

Support Protocol 2: Metabolic Labeling or Recombinant Proteins 16.11.3

Basic Protocol 2: Large-Scale Production of Recombinant Proteins 16.11.4

Basic Protocol 3: Purification of Recombinant Proteins Containing a
Polyhistidine (6xHis) Tag 16.11.6

Alternate Protocol: Purification of Recombinant Proteins Containing a
GSTTag 16.11.8

Reagents and Solutions 16.11.9

Commentary 16.11.10

EXPRESSION OF PROTEINS IN MAMMALIAN CELLS

Overview of Protein Expression in Mammalian Cells 16.12.1

Viral-Mediated Gene Transfer 16.12.1


Transient Expression 16.12.2

Stable DNA Transfection 16.12.3

AmpliFication of Transfected DNA 16.12.4

Expression Vectors 16.12.5

Choice of Expression System 16.12.5

Transient Expression of Proteins Using COS Cells 16.13.1

Basic Protocol 16.13.1

Commentary 16.13.3

Amplification Using CHO Cell Expression Vectors 16.14.1

Basic Protocol: Amplification Using Dihydrofolate Reductase 16.14.1

Alternate Protocol: Amplification by Cloning at Each Selective Step 16.14.7

Basic Protocol: Amplification Using Glutamine Synthetase 16.14.7

Reagents and Solutions 16.14.9

Commentary 16.14.10

EXPRESSION OF PROTEINS IN MAMMALIAN CELLS
USING VACCINIA VIRAL VECTORS

Overview of the Vaccinia Virus Expression System 16.15.1

Vaccinia Replication Cycle 16.15.1

Effects of Vaccinia Infection 16.15.2

Vaccinia Vector Expression System 16.15.3

Steps for Expression of Genes Using Vaccinia Vectors 16.15.3

Safety Precautions for Using Vaccinia 16.15.5


Preparation of Cell Cultures and Vaccinia Virus Stocks 16.16.1

Basic Protocol 1: Culture of Monolayer Cells 16.16.2

Basic Protocol 2: Culture of Cells in Suspension 16.16.3


Basic Protocol 3: Preparation of a Vaccinia Virus Stock 16.16.4

Support Protocol I: Titration of Vaccinia Virus Stocks by Plaque Assay 16.16.5

Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts 16.16.6

Basic Protocol 5: Preparation of an MVA Stock 16.16.8

Support Protocol 2: Titration of MVA Stocks by Immunostaining 16.16.9

Reagents and Solutions 16.16.10

Commentary 16.16.11

Generation of Recombinant Vaccinia Viruses 16.17.1

Basic Protocol I: Transfection of Infected Cells with a Vaccinia Vector 16.17.1

Support Protocol 1: Purification of Vaccinia Virus 16.17.5

Support Protocol 2: Isolation of Vaccinia Virus DNA 16.17.8

Basic Protocol 2: Selection and Screening of Recombinant Virus Plaques 16.17.9

Basic Protocol 3: Amplification of a Plaque 16.17.12

Basic Protocol 4: Live lmrnunostaining ofMVA Recombinants 16.17.14

Support Protocol 3: Coating Plates with Concanavalin A 16.17.15

Reagents and Solutions 16.17.16

Commentary 16.17.16

Characterization of Recombinant Vaccinia Viruses and
Their Products 16.18.1

Basic Protocol I: Detection of Vaccinia DNA Using PCR 16.18.1

Basic Protocol 2: Detection of Vaccinia DNA using Southern Blot
Hybridization 16.18.4

Basic Protocol 3: Detection of Vaccinia DNA Using Dot-Blot Hybridization 16.18.5

Alternate Protocol: Detection of Expressed Protein by a Dot-Blot Procedure 16.18.6

Basic Protocol 4: Detection of Expressed Protein Using Immunoblotting 16.18.7

Basic Protocol 5: Detection of Expressed Protein Using Immunoprecipitation 16.18.8

Reagents and Solutions 16.18.9


Commentary 16.18.10

Gene Expression Using the Vaccinia/T7 RNA Polymerase
Hybrid System 16.19.1

Basic Protocol 1: Lipsome-Mediated Transfection Following Recombinant
Vaccinia Virus (vTF7-3) Infection 16.19.1

Basic Protocol 2: Coinfection with Two Recombinant Vaccinia Viruses 16.19.4

Basic Protocol 3: Infection ofOST7-l Cells with a Single Virus 16.19.5

Basic Protocol 4: Gene Expression Using the VOTE System 16.19.6

Support Protocol: Detection of Expressed Protein Using Pulse Labeling 16.19.8

Commentary 16.19.9


Expression of Proteins Using Semliki Forest Virus Vectors 16.20.1

Strategic Planning: Choice of SFV Vector 16.20.1

Basic Protocol: Expression of Proteins from Recombinant SPV RNA
Using Electroporative Transfection 16.20.3

Alternate Protocol: Expression of Proteins from Recombinant SFV RNA
Using Liposome-Mediated Transfection 16.20.5

Support Protocols: Screening for Gene Expression Using P-Galactosidase 16.20.7

Screening of Cells for P-Galactosidase Activity 16.20.7

Screening of Cell Lysates for P-Galactosidase Activity 16.20.7

Basic Protocol: Expression of Protein from In Vivo-Packaged Recombinant
SFV Particles 16.20.8

Support Protocol: Determination of Recombinant Virus Titer 16.20.9

Support Protocol: Purification of SFV Particles 16.20.10

Reagents and Solutions 16.20.11

Commentary 16.20.13


SPECIALIZED EXPRESSION SYSTEMS

Inducible Gene Expression Using an Autoregulatory,
Tetracycline-Controlled System 16.21.1

Basic Protocol: Calcium Phosphate-Mediated Stable Transfection of
NIH3T3 Cells with pTet-tTAk and Tetracycline-Regulated Target Plasmids 16.21.1

Support Protocol: Analysis of Target Gene Protein Expression 16.21.6

Reagents and Solutions 16.21.7

Commentary 16.21.8

PREPARATION AND ANALYSIS
OF GLYCOCONJUGATES

INTRODUCTION 17.0.5

Overview of Glycoconjugate Analysis 17.0.5

Choice of Techniques 17.0.7

Stereochemistry and Diagrammatic Representation of Saccharides 17.0.9

Glossary 17.0.12

SPECIAL CONSIDERATIONS OF GLYCOCONJUGATES AND
THEIR PURIFICATION

Special Considerations for Glycoproteins and Their Purification 17.1.2

Basic Protocol: Con A-Sepharose Affinity Chromatography 17.1.5

Support Protocol: Pilot Study 17.1.7

Alternate Protocol: WGA-Agarose Affinity Chromatography 17.1.8

Special Considerations for Protcoglycans and Glycosaminoglycans
and Their Purification 17.2.1

Basic Protocol: High-Salt / Detergent Extraction 17.2.1

Alternate Protocol: Alkali Extraction 17.2.2

Basic Protocol: Anion-Exchange Chromatography 17.2.3

Alternate Protocol: CPC/Ethanol Precipitation 17.2.5

Special Considerations for Glycolipids and Their Purification 17.3.1

Basic Protocol: Extraction and Purification of Glycolipids 17.3.2

Support Protocol: Preparation of Sep-Pak 018 Cartridges 17.3.6

Basic Protocol: Preparation of Gangliosides 17.3.7

Alternate Protocol: Rapid Purification of Gangliosides 17.3.8

DETECTION OF SACCHARIDES ON GLYCOCONJÜGATES

Metabolic Radiolabeling of Animal Cell Glycoconjugates 17.4.2

Basic Protocol: Steady-State Labeling with Radioactive Precursors 17.4.2

Alternate Protocol: Pulse or Pulse-Chase Labeling 17.4.4

Alternate Protocol: Sequential Pulse or Pulse-Chase Labeling 17.4.7

Support Protocol: Preparation of Multiply Deficient Medium 17.4.8

Chemical Labeling of Carbohydrates by Oxidation and
Sodium Borohydride Reduction 17.5.1

Basic Protocol: Radiolabeling Oligosaccharides After Oxidation 17.5.1

Alternate Protocol: Radiolabeling After Galactose Oxidase Treatment 17.5.3

Alternate Protocol: Fluorescence Labeling Oligosaccharides After
Periodate or Galactose Oxidase Treatment 17.5.4

Basic Protocol: Radiolabeling Free Oligosaccharides 17.5.5

Alternate Protocol: Radiolabeling O-Glycan Oligosaccharides 17.5.6

Analysis of Saccharide Structure and Function Using.
Glycosyltransferases 17.6.1

Basic Protocol: Detection of O-Linked A N -AcetyIglucosamine 17.6.1

Support Protocol: Autogalactosylation of Galactosyltransferase 17.6.3

Support Protocol: Assay of Galactosyltransferase Activity 17.6.4

Basic Protocol: Labeling Terminal Galpl-4GlcNAc Residues 17.6.6

Lectin Analysis of Proteins Blotted onto Filters 17.7.1

Detection of Glycophospholipid Anchors on Proteins 17.8.1

Basic Protocol: Extraction and Partitioning of Total Proteins 17.8.3

Alternate Protocol: Partitioning of Isolated Proteins with Triton X-114 17.8.4

Support Protocol: Precondensation of Triton X-114 Detergent 17.8.4

Basic Protocol: 1 dentification of GPI-Anchored Proteins by PI-PLC 17.8.5

Alternate Protocol: Identification of GPI Anchorage by Phospholipase 17.8.5

Support Protocol: Detection of Products with Anti-CRD Antibody 17.8.7

Basic Protocol: Nitrous Acid Cleavage of GPI-Anchored Proteins 17.8.7

Support Protocol: Separation of Lipid Moiety 17.8.8

Basic Protocol: Base Hydrolysis of Radiolabeled Proteins 17.8.8

Direct Chemical Analysis of Glycoconjugates for Carbohydrates 17.9.1

Basic Protocol 1: Phenol-Sulfuric Acid Assay for Hexoses and Pentoses 17.9.1

Basic Protocol 2: Ferric Orcinol Assay for Sialic Acids 17.9.3

Basic Protocol 3: MBTH Assay for Hexosamines and Acetylhexosamines 17.9.5

Assays for Uronic Acids 17.9.8

Basic Protocol 4: Carbazole Assay for Uronic Acids 17.9.8

Alternate Protocol: Afcto-Hydroxybiphenyl Assay for Uronic Acids 17.9.9


Inhibition of N-Linked Glycosylation 17.10.1

Basic Protocol 17.10.1

Support Protocol: Acetone Precipitation 17.10.6


Inhibition of Glycolipid Biosynthesis 17.10.10

Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and
Inhibitors of Glycoprotcin and Protcoglycan Assembly 17.11.1

Basic Protocol 1: Xyloside Initiation of Gag Synthesis 17,11.1

Basic Protocol 2: Initiation of Oligosaccharide Synthesis 17.11.2

RELEASE OF SACCHARIDES FROM GLYCOCONJUGATES

Sialidases 17.12.2

Basic Protocol: Sialidase Treatment of Purified Glycoproteins 17.12.2

Alternate Protocol: Sialidase Treatment of Intact Cells 17.12.5


Endoglycosidase and Glycoamidase Release of
N-Linked Oligosaccharides 17.13.1

Basic Protocol: Endoglycosidase H Digestion 17.13.3


Basic Protocol: Endoglycosidase F2 Digestion 17.13.4

Basic Protocol: Peptide:N-Glycosidase F Digestion 17.13.5

Support Protocol: Estimating the Number of N-Linked Chains 17.13.6

Support Protocol: Digestion and Recovery of Released N-Linked Chains 17.13.8


Analysis of Glycosaminoglycans with Polysaccharide Lyascs 17.13.17

Overview of HeparinLyases 17.13.18

HeparinLyasel 17.13.19

HeparinLyasell 17.13.23

HeparinLyaselll 17.13.23

Overview of Chondroitin Sulfate Lyases 17.13.23

ChondroitinABCLyase 17.13.23

Chondroitin AC Lyase 17.13.26

Chondroitin B Lyase 17.13.26

Hyaluronate Lyase 17.13.27
Preparation of Glycopeptides 17.14.1

Basic Protocol: Extensive Proteolytic Digestion of Glycoproteins 17.14.1

Basic Protocol: Proteolytic Digestion of Purified Glycoproteins 17.14.4

Detection of Individual Glycosylation Sites on Glycoproteins 17.14.10

Basic Protocol: Fractionation ofGlycopeptides by Reversed-Phase HPLC 17.14.10

Support Protocol: Endoglycosidase Digestion of Purified Glycopeptides 17.14.12

(beta)-Elimination for Release of O- Linked Glycosaminoglycans
from Proteoglycans 17.15.1

(beta)-Elimination for Release of O-GalNAc-Linked Oligosaccharides
from Glycoproteins and Glycopeptides 17.15.4

Acid Hydrolysis for Release of Monosaccharides 17.16.1

Basic Protocol 1: Mild Acid Hydrolysis for Release of Fucose Residues 17.16.2

Basic Protocol 2: Release of Sialic Acids (Excluding 4-O-Acetylated) 17.16.3

Alternate Protocol: Release of 4-O-Acetylated Sialic Acids 17.16.5

Basic Protocol 3: Strong Acid Hydrolysis for Quantitative Release 17.16.6


Enzymatic Release of Oligosaccharides from Glycolipids 17.17.1

Basic Protocol: Use of Endoglycoceramidase for Selective Cleavage 17.17.1

Endo-(beta)-Galactosidases and Keratanase 17.17.6

Endo-(beta)-Galactosidase from E. Freundii 17.17.6

Endo-(beta)-Galactosidase from Bacteroides Fragilis 17.17.10

Endo-(beta)-Galactosidase from Flavobacterium Keratolyticus 17.17.10

Blood-Group Type A and B-Specific Endo-p-Galactosidase 17.17.10

Endo-(beta)-Galactosidase DII from Diplococcus Pneumoniae 17.17.10

Endo-p-Galactosidase C from Clostridium Perfringens 17.17.11

Keratanase from Pseudomonas Sp. 17.17.12

Endo-(beta)-Galactosidase Specific to Chondroitin Sulfate-Core Link 17.17.12

ANALYSIS OF SACCHARIDES RELEASED FROM GLYCOCONJUGATES

Analysis of Monosaccharides 17.18.1

Basic Protocol 1: BCA Assay for Reducing Sugars 17.18.1

Basic Protocol 2: Assays for Free Hexosamines 17.18.4

Basic Protocol 3: Thiobarbituric Acid Assay for Sialic Acids 17.18.8

Basic Protocol 4: DMB Assay for Sialic Acids with HPLC Detection 17.18.15

Total Compositional Analysis by High-Performance Liquid
Chromatography or Gas-Liquid Chromatography 17.19.1

Strategic Planning 17.19.1

Compositional Analysis of Free Monosaccharides by HPAEC-PAD 17.19.3

Basic Protocol 1: Mixtures of Monosaccharides and Hexosamines 17.19.4

Basic Protocol 2: Mixtures of Monosaccharides, Hexosamines, and
Uronic Acids 17.19.8

Basic Protocol 3: Analysis of Mixtures of Sialic Acids 17.19.9

Alternate Protocol 1: Compositional Analysis of Free Sialic Acids 17.19.12

Basic Protocol 4: Compositional Analysis by GLC-FID 17.19.14

Support Protocol 1: Quantitative Release by Methanolysis 17.19.16

Support Protocol 2: Preparation of Volatile Derivatives of Free Glycoses 17.19.17

Support Protocol 3: Preparation of Volatile Derivatives 17.19.19

Support Protocol 4: Preparation of Monosaccharides Standard Solutions 17.19.19
Composition of Labeled Monosaccharides from Glycosammoglycans 17.19.26

Basic Protocol 17.19.26

Analysis of Oligosaccharide Negative Charge by Anion-Exchange
Chromatography 17.20.1

Basic Protocol: Separation and Analysis of Anionic Oligosaccharides by
QAE-Sephadex Chromatography with Stepwise Elution 17.20.1

Alternate Protocol: Separation and Analysis of Anionic Oligosaccharides
by QAE-Sephadex Chromatography with Gradient Elution 17.20.3

Support Protocol: Detection and Removal of Phosphodi- or Monoesters 17.20.4

HPLC Methods for the Fractionation and Analysis of Negatively
Charged Oligosaccharides and Gangliosides 17.21.1

Basic Protocol 1: Fractionation of Released N-Linked Oligosaccharides 17.21.1

Basic Protocol 2: Fractionation of Gangliosides on DEAE HPLC Columns 17.21.3

Fractionation and Analysis of Neutral Oligosaccharides by HPLC 17.21.9

Basic Protocol: Separation of Neutral Oligosaccharides on Bonded Amine
HPLC 17.21.9

Support Protocol: Sodium Borohydride Reduction and Desalting 17.21.10

Nitrous Acid Degradation of Glycosaminoglycans 17.22.1

Basic Protocol 17.22.1

Analysis of Disaccharides and Tetrasaccharides Released from
Glycosaminoglycans 17.22.6

Basic Protocol 1: Analysis ofDi- and Oligosaccharides by Paper
Chromatography and Paper Electrophoresis 17.22.6

Basic Protocol 2: Analysis of Di- and Oligosaccharides by HPLC 17.22.8

Support Protocol 1: Borohydride Reduction of Alkali-Labile
Disaccharides 17.22.12

Support Protocol 2: Scintillation Counting of Fractions from HPLC 17.22.13

Support Protocol 3: Calculations for Quanitation of Disaccharides 17.22.13

Analysis of Sulfate Esters by Solvolysis or Hydrolysis 17.23.1

Basic Protocol 1: Solvolysis for Release of Sulfate Esters from Glycans 17.23.1

Support Protocol 1: Monitoring Solvolysis Reaction 17.23.2

Alternate Protocol 1: Scale-Up of Solvolysis 17.23.3

Basic Protocol 2: Acid Hydrolysis for Release of Sulfate Esters 17.23.4

Alternate Protocol 2: Base Hydrolysis for Release of Sulfate Esters 17.23.6

ANALYSIS OF PROTEIN
PHOSPHORYLATION

INTRODUCTION 18.0.1

Overview of Protein Phosphorylation 18.1.1

History 18.1.1

Labeling Studies 18.1.1

Sites of Phosphorylation 18.1.2

Detection of unlabeled Phosphoamino Acids 18.1.2

Protein Kinases 18.1.3

Protein Phosphatases 18.1.3

Literature Cited 18.1.4


Labeling Cultured Cells with (32)Pi and Preparing
Cell Lysates for Immunoprecipitation 18.2.1

Basic Protocol: Labeling Cultured Cells with (32)Pi and Lysis
using Mild Detergent 18.2.1

Alternate Protocol: Lysis of Cells by Boiling in SDS 18.2.4

Reagents and Solutions 18.2.5

Commentary 18.2.6

Phosphoamino Acid Analysis 18.3.1

Basic Protocol: Acid Hydrolysis and Two-Dimensional Electrophoretic
Analysis of Phosphoamino Acids 18.3.1

Alternate Protocol: Alkali Treatment to Enhance Detection of Tyr- and
Thr-Phosphorylated Proteins Blotted onto Filters 18.3.5

Reagents and Solutions 18.3.6

Commentary 18.3.7

Analysis of Phosphorylation of Unlabeled Proteins 18.4.1

Basic Protocol 1: Immunoblotting with Anti-Phosphotyrosine Antibodies and
Detectiion Using {125 I }Protein A 18.4.1

Alternate Protocol: Detection of Bound Antibodies by Enhanced
Cherniluminescence(ECL) 18.4.3

Basic Protocol 2: Identification of Phosphorylated Proteins by Phosphatase
Digestion 18.4.4

Reagents and Solutions 18.4.5

Commentary 18.4.5

Detection of Phosphorylation by Enzymatic Techniques 18.5.1

Basic Protocol 1: Digestion ofPhosphoproteins with Nonspecific Acid
Phosphatases 18.5.2

Alternate Protocol 1: Digestion of Phosphoproteins with Nonspecific Alkaline
Phosphatase 18.5.3

Basic Protocol 2: Digestion of Phosphoproteins with Protein Serine/Threonine
Phosphatases 18.5.4

Alternate Protocol 2: Digestion of Phosphoproteins with Protein Tyrosine
Phosphatases 18.5.4

Support Protocol: Measurement and Identification of Released (32) P 18.5.5

Commentary 18.5.6

Production of Antibodies That Recognize Specific Tyrosine-
Phosphorylated Peptides 18.6.1

Basic Protocol 1: Production of Polyclonal Anti-Phosphopeptide Antibodies 18.6.2

Basic Protocol 2: Production of Monoclonal Anti-Phosphopeptide Antibodies 18.6.6

Support Protocol 1: Synthesis of Peptides 18.6.11

Support Protocol 2: Coupling of Peptides to Affi-Gel 10 Affinity Matrix 18.6.11

Support Protocol 3: Coupling ofPhosphotyrosine to Affi-Gel 10 Affinity Matrix 18.6.13

Reagents and Solutions 18.6.14

Commentary 18.6.14

Assays of Protein Kinases Using Exogenous Substrates 18.7.1

Strategic Planning 18.7.1

Basic Protocol 1: Assay for Cyclic Nucleotide-Dependent Protein Kinases 18.7.4

Basic Protocol 2: Assay for Protein Kinase C Isoforms 18.7.6

Basic Protocol 3: Assay for Casein Kinases Using p-Casein 18.7.7

Alternate Protocol: Assay for Casein Kinases Using a Peptide Substrate 18.7.8

Basic Protocol 4: Assay for Ca(2+)/Calmodulin-Dependent Kinases 18.7.9

Basic Protocol 5: Assay for Tyrosine Kinases 18.7.11

Basic Protocol 6: In-Gel Protein Kinase Assays 18.7.12

Support Protocol 1: Preparing a Cell Lysate for Kinase Assays 18.7.14

Support Protocol 2: TCA Precipitation to Determine Incorporation
of Radioactivity 18.7.14

Support Protocol 3: Adsorption onto P81 Phosphocellulose Paper 18.7.15

Support Protocol 4: Electrophoretic Analysis of Phosphorylation 18.7.17

Reagents and Solutions 18.7.18

Commentary 18.7.20

Permeabilization Strategies to Study Protein Phosphorylation 18.8.1

Basic Protocol 1: Analysis of Protein Phosphorylation in Permeabilized Cells 18.8.2

Intact Cell Sample Preparation for Electrophoretic Analysis of Protein
Phosphorylation 18.8.6

Alternate Protocol 1: Intact Cell Sample Preparation for SDS-PAGE 18.8.6

Alternate Protocol 2: Intact Cell Sample Preparation for Isoelectric
Focusing 18.8.7

Basic Protocol 2: Analysis of Protein Phosphorylation Using Isolated
Subcellular Fractions 18.8.7

Organelle Sample Preparation for Electrophoretic Analysis of Protein
Phosphorylation 18.8.9

Alternate Protocol 3: Organelle Sample Preparation for SDS-PAGE 18.8.9

Alternate Protocol 4: Organelle Sample Preparation for Isoelectric
Focusing 18.8.9

Alternate Protocol 5: Direct Analysis ofCytosolic or Membrane-Bound
Kinases 18.8.10

Support Protocol 1: Determination of Specific Radioactivity 18.8.11

Support Protocol 2: Determination of the Specific Activity of the (gamma) -Phosphate
of (32P)ATP 18.8.14

Reagents and Solutions 18.8.16

Commentary 18.8.18

INFORMATICS FOR
MOLECULAR BIOLOGISTS

INTRODUCTION 19.0.3

Internet Basics for Biologists 19.1.1

Internet Basics 19.1.1

Connecting to the Internet 19.1.1

Electronic Mail 19.1.3

File Transfer Protocol 19.1.8

Gopher 19.1.12

The World Wide Web 19.1.13

Note 19.1.18

Literature Cited 19.1.18

Sequence Databases: Information Retrieval and Data Submission 19.2.1

Introduction to Entrez 19.2.3

Data Submission: General Considerations 19.2.5

Submitting a Sequence to the Nucleotide Database 19.2.6

Submitting an update or Correction to an Existing GenBank Entry 19.2.10

Submitting EST, STS, or GSS Data 19.2.10

Submitting Mapping Data 19.2.10

Submitting High-Throughput Genome Sequences (HTGS) 19.2.11

Conclusion 19.2.11

Literature Cited 19.2.11

Internet Resources 19.2.12

Appendix: Sample GenBank Records 19.2.14


Sequence Similarity Searching Using the BLAST Family of Programs 19.3.1

Obtaining BLAST Documentation and Help 19.3.1

Submitting Sequences for Database Homology Searching 19.3.2

Basic Protocol 1: Submitting a Sequence to BLAST Using the Electronic
Mail Server 19.3.3

Basic Protocol 2: Submitting a Sequence to BLAST Using the Command-
Line Client Program 19.3.4


Basic Protocol 3: Submitting a Sequence Using NetBLAST Graphical Client
Program 19.3.5

Basic Protocol 4: Submitting a Sequence Using the WWW Interface 19.3.5

Example 1: Using BLASTN to Search a Nucleotide Sequence Database with
a DNA Sequence 19.3.6

Example 2: Using BLASTX to Search a Protein Sequence Database with
Open Reading Frames Derived from Conceptural Translations of a DNA
Query Sequence 19.3.10

Example 3: Using BLASTP to Search a Protein Sequence Database with a
Protein Sequence Query Using the Filter Option to Mask Low-
Complexity Sequences 19.3.14

Example 4: Using the WWW Interface 19.3.19

Example 5: Using TBLASTN to Search a Translated Nucleotide Database
with a Protein Sequence 19.3.20

Example 6: Using the NetBLAST Graphical Client Program 19.3.25

Example 7: Using TBLASTX to Search Open Reading Frames Derived
from Conceptual Translations of a Nucleotide Sequence Database Using
Open Reading Frames Derived from a Nucleotide Sequence Query 19.3.26

Commentary 19.3.29

ANALYSIS OF PROTEIN
INTERACTIONS

INTRODUCTION 20.0.1

Interaction Trap/Two-Hybrid System to Identify Interacting Proteins 20.1.1

Basic Protocol 1: Characterizing a Bait Protein 20.1.3

Basic Protocol 2: Performing an Interactor Hunt 20.1.11

Alternate Protocol: Rapid Screen for Interaction Trap Positives 20.1.20

Support Protocol 1: Preparation of Protein Extracts for Immunoblot Analysis 20.1.22

Support Protocol 2: Preparation of Sheared Salmon Sperm Carrier DNA 20.1.23

Support Protocol 3: Yeast Colony Hybridization 20.1.24

Support Protocol 4: Microplate Plasmid Rescue 20.1.26

Support Protocol 5: Additional Specificity Screening 20.1.27

Reagents and Solutions 20.1.28

Commentary 20.1.28

Affinity Purification of Proteins Binding to GST Fusion Proteins 20.2.1

Strategic Planning 20.2.1

Basic Protocol: GST Fusion Protein-Affinity Purification 20.2.3

Support Protocol: Preparation ofE. coli Extract 20.2.5

Reagents and Solutions 20.2.6

Commentary 20.2.7

Phage-Based Expression Cloning to Identify Interacting Proteins 20.3.1

Strategic Planning 20.3.1

Basic Protocol: Interaction Cloning 20.3.2

Reagents and Solutions 20.3.6

Commentary 20.3.6

Surface Plasmon Resonance for Measurements of Biological Interest 20.4.1

Basic Protocol 1: SPR Using BIAcore Chips 20.4.2

Basic Protocol 2: SPR Using NTA-SAM Chips 20.4.3

Commentary 20.4.4