Preface ................................................. xxvii
Contributors ............................................. xxxi
13. Saccharomyces cerevisiae ................................. 13-1
13.1. Preparation of Yeast Media ......................... 13-2
Liquid Media ....................................... 13-2
Solid Media ........................................ 13-5
Strain Storage and Revival ......................... 13-7
Basic Protocol 1: Preparation and Inoculation of
Frozen Stocks ...................................... 13-7
Alternate Protocol: Preparation and Inoculation
of Slants .......................................... 13-7
Basic Protocol 2: Mailing and Reviving Strains ..... 13-8
13.2. Growth and Manipulation of Yeast ................... 13-8
Basic Protocol 1: Growth in Liquid Media ........... 13-8
Basic Protocol 2: Growth on Solid Media ............ 13-9
Basic Protocol 3: Determination of Cell Density .... 13-9
Basic Protocol 4: Determination of Phenotype by
Replica Plating .................................... 13-9
Basic Protocol 5: Determination of Mating Type ..... 13-9
Strain Construction and Tetrad Analysis ........... 13-10
Basic Protocol 6: Diploid Construction ............ 13-11
Basic Protocol 7: Sporulation of Diploid Cells .... 13-11
Basic Protocol 8: Preparation and Dissection
of Tetrads ........................................ 13-12
Support Protocol: Preparation of Dissecting
Needles ........................................... 13-15
Alternate Protocol: Random Spore Analysis ......... 13-15
13.3. Genome-Wide Transposon Mutagenesis in Yeast ....... 13-17
Strategic Planning ................................ 13-17
Basic Protocol: Generating Yeast Mutants from
mTn-Mutagenized Library DNA ....................... 13-17
Support Protocol 1: Vectorette Polymerase Chain
Reaction .......................................... 13-20
Support Protocol 2: Epitope Tagging of
mTn-Mutagenized Gene Products ..................... 13-21
13.4. Yeast Vectors and Assays for Expression of
Cloned Genes ...................................... 13-23
Basic Protocol 1: Construction of lacZ Fusion
Vectors for Studying Yeast Gene Regulation ........ 13-23
Basic Protocol 2: Assay for β-Galactosidase in
Liquid Cultures ................................... 13-23
Alternate Protocol: Screening for β-
Galactosidase-Expressing Yeast Colonies
Using a Filter Lift Assay ......................... 13-24
13.5. Introduction of DNA into Yeast Cells .............. 13-25
Basic Protocol: Transformation Using
Lithium Acetate ................................... 13-25
Alternate Protocol: Transformation by
Electroporation ................................... 13-27
Support Protocol: Preparation of Single-Stranded
High-Molecular-Weight Carrier DNA ................. 13-28
13.6. Cloning Yeast Genes by Complementation ............ 13-29
Basic Protocol: Cloning the Gene .................. 13-29
13.7. Manipulation of Plasmids from Yeast Cells ......... 13-31
Basic Protocol 1: Segregation of Plasmids from
Yeast Cells ....................................... 13-31
Basic Protocol 2: Plasmid Shuffling ............... 13-31
Basic Protocol 3: Plasmid Gap Repair for
Localized Mutagenesis and Allele Repair ........... 13-33
13.8. Manipulation of Cloned Yeast DNA .................. 13-35
Basic Protocol 1: Integrative Transformation ...... 13-35
Gene Replacement Techniques ....................... 13-35
Basic Protocol 2: Integrative Disruption .......... 13-36
Basic Protocol 3: One-Step Gene Disruption ........ 13-36
Alternate Protocol 1: PCR-Mediated One-Step
Gene Disruption ................................... 13-37
Basic Protocol 4: Transplacement 13-38
Basic Protocol 5: Creating Modified Genes by
One-Step Integrative Replacement .................. 13-40
Alternate Protocol 2: Creating Modified Genes by
Transplacement .................................... 13-41
Basic Protocol 6: Creation of Conditional
Alleles by Copper-Inducible Double-Shutoff
Procedure ......................................... 13-41
13.9. Preparation of Yeast DNA .......................... 13-43
Basic Protocol: Rapid Isolation of Plasmid DNA
from Yeast ........................................ 13-44
Alternate Protocol: Rapid Isolation of Yeast
Chromosomal DNA ................................... 13-44
13.10.Preparation of Yeast RNA .......................... 13-45
Basic Protocol: Preparation of Yeast RNA by
Extraction with Hot Acidic Phenol ................. 13-45
Alternate Protocol 1: Preparation of RNA Using
Glass Beads ....................................... 13-46
Alternate Protocol 2: Preparation of
Poly(A)+ RNA ...................................... 13-47
13.11.Preparation of Protein Extracts from Yeast ........ 13-47
Basic Protocol: Spheroplast Preparation
and Lysis ......................................... 13-48
Support Protocol: Nuclei Preparation by
Differential Centrifugation ....................... 13-49
Alternate Protocol 1: Cell Disruption Using
Glass Beads ....................................... 13-50
Alternate Protocol 2: Cell Disruption Using
Liquid Nitrogen ................................... 13-50
14. In Situ Hybridization and Immunohistochemistry ........... 14-1
14.1. Fixation, Embedding, and Sectioning of Tissues,
Embryos, and Single Cells .......................... 14-2
Basic Protocol: Paraformaldehyde Fixation and
Paraffin Wax Embedding of Tissues and Embryos ...... 14-2
Alternate Protocol: Fixation of Suspended and
Cultured Cells ..................................... 14-4
Support Protocol 1: Perfusion of Adult Mice ........ 14-4
Support Protocol 2: Sectioning Samples in
Wax Blocks ......................................... 14-6
Support Protocol 3: Preparation of Coated
Slides ............................................. 14-7
14.2. Cryosectioning ..................................... 14-8
Basic Protocol: Specimen Preparation and
Sectioning ......................................... 14-8
Support Protocol 1: Fixation of Cryosections for
In Situ Hybridization ............................. 14-10
Support Protocol 2: Tissue Fixation and Sucrose
Infusion .......................................... 14-12
14.3. In Situ Hybridization to Cellular RNA ............. 14-13
Basic Protocol: Hybridization Using Paraffin
Sections and Cells ................................ 14-13
Alternate Protocol: Hybridization Using
Cryosections ...................................... 14-16
Support Protocol 1: Synthesis of 35S-Labeled
Riboprobes ........................................ 14-18
Support Protocol 2: Synthesis of MS-Labeled
Double-Stranded DNA Probes ........................ 14-19
14.4. Detection of Hybridized Probe ..................... 14-20
Basic Protocol 1: Film Autoradiography ............ 14-20
Basic Protocol 2: Emulsion Autoradiography ........ 14-20
Support Protocol: Preparation of Diluted
Emulsion for Autoradiography ...................... 14-21
14.5. Counterstaining and Mounting of Autoradiographed
In Situ Hybridization Slides ...................... 14-22
Basic Protocol: Giemsa Staining ................... 14-22
Alternate Protocol 1: Hematoxylin/Eosin
Staining .......................................... 14-23
Alternate Protocol 2: Toluidine Blue Staining ..... 14-24
Alternate Protocol 3: Hoechst Staining ............ 14-24
14.6. Immunohistochemistry .............................. 14-25
Basic Protocol 1: Immunofluorescent Labeling of
Cells Grown as Monolayers ......................... 14-25
Alternate Protocol 1: Immunofluorescent Labeling
of Suspension Cells ............................... 14-28
Basic Protocol 2: Immunofluorescent Labeling of
Tissue Sections ................................... 14-28
Alternate Protocol 2: Immunofluorescent
Labeling Using Streptavidin-Biotin Conjugates ..... 14-29
Alternate Protocol 3: Immunogold Labeling of
Tissue Sections ................................... 14-30
Alternate Protocol 4: Immunoperoxidase Labeling
of Tissue Sections ................................ 14-30
Alternate Protocol 5: Immunofluorescent
Double-Labeling of Tissue Sections ................ 14-31
14.7. In Situ Hybridization and Detection Using
Nonisotopic Probes ................................ 14-32
Basic Protocol: Fluorescence In Situ
Hybridization ..................................... 14-32
Amplification of Hybridization Signals ............ 14-34
Support Protocol 1: Amplification of
Biotinylated Signals .............................. 14-34
Support Protocol 2: Amplification of Signals
from Digoxigenin-Labeled Probes ................... 14-35
Enzymatic Detection of Nonisotopically Labeled
Probes ............................................ 14-36
Alternate Protocol 1: Enzymatic Detection Using
Horseradish Peroxidase ............................ 14-37
Alternate Protocol 2: Enzymatic Detection Using
Alkaline Phosphatase .............................. 14-38
14.8. In Situ Polymerase Chain Reaction and
Hybridization to Detect Low-Abundance Nucleic
Acid Targets ...................................... 14-39
Strategic Planning ................................ 14-39
Basic Protocol 1: In Situ PCR (ISPCR)
Amplification of DNA and RNA Targets with
In Situ Reverse Transcription for RNA ............. 14-42
Alternate Protocol: One-Step Reverse
Transcription and Amplification ................... 14-45
Basic Protocol 2: Hybridization and Detection of
ISPCR-Amplified Target Material ................... 14-46
Support Protocol 1: Preparation of AES-Subbed
Slides ............................................ 14-50
Support Protocol 2: Preparation of Specimens on
Slides for ISPCR .................................. 14-50
Support Protocol 3: Labeling Oligonucleotide
Probes Using MP ................................... 14-51
14.9. Whole-Mount In Situ Hybridization and Detection
of RNAs in Vertebrate Embryos and Isolated
Organs ............................................ 14-52
Basic Protocol 1: Whole-Mount In Situ
Hybridization with Mouse or Chicken
Embryos and Organs ................................ 14-52
Basic Protocol 2: Enzymatic Detection of RNA
Hybrids in Mouse and Chicken Embryos and Organs ... 14-54
Alternate Protocol 1: Whole-Mount In Situ
Hybridization with Xenopus Embryos ................ 14-57
Alternate Protocol 2: Enzymatic Detection of
RNA Hybrids in Xenopus Embryos .................... 14-58
Support Protocol 1: Synthesis of Digoxigenin-
Labeled RNA Probes ................................ 14-59
Support Protocol 2: Preabsorption of Fab
Fragments with Embryonic Powder ................... 14-60
14.10.Principles and Application of Fluorescence
Microscopy ........................................ 14-61
Fluorescent Molecular Probes ...................... 14-62
Filters and Filter Sets ........................... 14-63
Multiband Filters and Multidye Fluorescence ....... 14-64
Light Sources ..................................... 14-64
Microscope Objectives ............................. 14-65
Image Resolution and the Point-Spread
Function (PSF) .................................... 14-65
Fluorescence Microscopy of Living Cells ........... 14-66
Immunolabeling: General Steps for Labeling Fixed
Cells and Tissues ................................. 14-67
14.11.Basic Confocal Microscopy ......................... 14-71
The Basis of Optical Sectioning ................... 14-71
Types of Confocal Microscopes ..................... 14-74
Practical Guidelines .............................. 14-76
Internet Resources ................................ 14-81
14.12.Measurement of In Situ Hybridization .............. 14-82
Basic Protocol: Determining the Distribution
of Radiolabeled Heteroduplexes in In Situ
Hybridizations by Phosphor Storage Imaging ........ 14-82
Support Protocol: Producing a Reference System .... 14-85
14.13.Morphological, Biochemical, and Flow
Cytometric Assays of Apoptosis .................... 14-86
Basic Protocol 1: Microscopic Quantitation of
Apoptotic Index and Cell Viability Using Vital
and Fluorescent Dyes .............................. 14-86
Basic Protocol 2: Determination of Apoptosis
Using Sub-G0/Gi DNA Peak .......................... 14-88
Basic Protocol 3: Flow Cytometric Quantitation
of Apoptotic Cells Using TUNEL .................... 14-89
Basic Protocol 4: In Situ Detection of Apoptotic
Cells in Tissue Sections by TUNEL ................. 14-91
14.14.Whole-Mount Histochemical Detection of
β-Galactosidase Activity .......................... 14-92
Basic Protocol: Whole-Mount Staining and
Histochemical Detection of β-Galactosidase
Activity .......................................... 14-92
Alternate Protocol: Preparing Thick Sections of
Large Tissues for P-Galactosidase Staining ........ 14-94
Support Protocol 1: Storage and Tissue Clearing ... 14-94
Support Protocol 2: Paraffin Embedding,
Sectioning, and Counter-Staining .................. 14-95
15. The Polymerase Chain Reaction ............................ 15-1
15.1. Enzymatic Amplification of DNA by PCR: Standard
Procedures and Optimization ........................ 15-3
Basic Protocol: Enzymatic Amplification of DNA
by PCR: Standard Procedures and Optimization ....... 15-3
15.2. Direct DNA Sequencing of PCR Products ............. 15-10
Basic Protocol 1: Generating Single-Stranded
Products for Dideoxy Sequencing by Asymmetric
PCR ............................................... 15-10
Alternate Protocol 1: Generating Single-Stranded
Template for Dideoxy Sequencing by Single-Primer
Reamplification ................................... 15-11
Alternate Protocol 2: Preparing Double-Stranded
PCR Products for Dideoxy Sequencing ............... 15-11
Alternate Protocol 3: Generating Single-Stranded
Template for Dideoxy Sequencing by X Exonuclease
Digestion of Double-Stranded PCR Products ......... 15-12
Basic Protocol 2: Labeling PCR Products for
Chemical Sequencing ............................... 15-13
Alternate Protocol 5: Genomic Sequencing of PCR
Products .......................................... 15-14
15.3. Ligation-Mediated PCR for Genomic Sequencing
and Footprinting .................................. 15-15
Basic Protocol: Ligation-Mediated Single-Sided
PCR ............................................... 15-15
Support Protocol 1: Preparation of Genomic DNA
from Monolayer Cells for DMS Footprinting ......... 15-19
Support Protocol 2: Preparation of Genomic DNA
from Suspension Cells for DMS Footprinting ........ 15-23
Support Protocol 3: Preparation of Genomic DNA
for Chemical Sequencing ........................... 15-24
15.4. Molecular Cloning of PCR Products ................. 15-25
Basic Protocol: Generation of T-A Overhangs ....... 15-26
Alternate Protocol 1: Generation of Half-Sites .... 15-27
15.5. Enzymatic Amplification of RNA by PCR (RT-PCR) .... 15-29
Basic Protocol: PCR Amplification of RNA Under
Optimal Conditions ................................ 15-29
Alternate Protocol 1: Avoiding Lengthy
Coprecipitation and Annealing Steps ............... 15-31
Alternate Protocol 2: Introducing cDNA Directly
into the Amplification Step ....................... 15-31
Support Protocol: Rapid Preparation of Crude
RNA ............................................... 15-31
15.6. cDNA Amplification Using One-Sided (Anchored)
PCR ............................................... 15-32
Basic Protocol 1: Amplification of Regions
Downstream (3') of Known Sequence ................. 15-32
Basic Protocol 2: Amplification of Regions
Upstream (5') of Known Sequence ................... 15-36
15.7. Quantitation of Rare DNAs by PCR .................. 15-38
Basic Protocol: Quantitation of Rare DNA by
PCR ............................................... 15-38
16. Protein Expression ....................................... 16-1
16.1. Overview of Protein Expression in E. coli .......... 16-2
General Strategy for Gene Expression in
E. coli ............................................ 16-3
Specific Expression Scenarios ...................... 16-3
Troubleshooting Gene Expression .................... 16-4
16.2. Expression Using the T7 RNA Polymerase/Promoter
System ............................................. 16-5
Basic Protocol: Expression Using the Two-Plasmid
System ............................................. 16-6
Alternate Protocol 1: Selective Labeling of
Plasmid-Encoded Proteins ........................... 16-7
Alternate Protocol 2: Expression by Infection
with M13 Phage mGP1-2 .............................. 16-8
16.3. Introduction to Expression by Fusion Protein
Vectors ............................................ 16-9
Solubility of the Expressed Protein ............... 16-10
Stability of the Expressed Protein ................ 16-10
Cleavage of Fusion Proteins to Remove
the Carrier ....................................... 16-11
16.4. Enzymatic and Chemical Cleavage of Fusion
Proteins .......................................... 16-12
Basic Protocol 1: Enzymatic Cleavage of Fusion
Proteins with Factor Xa ........................... 16-12
Support Protocol: Denaturing a Fusion Protein
for Factor Xa Cleavage ............................ 16-13
Alternate Protocol 1: Enzymatic Cleavage of
Fusion Proteins with Thrombin ..................... 16-13
Alternate Protocol 2: Enzymatic Cleavage of
Matrix-Bound GST Fusion Proteins .................. 16-14
Alternate Protocol 3: Enzymatic Cleavage
Fusion Proteins with Enterokinase ................. 16-15
Basic Protocol 2: Chemical Cleavage of Fusion
Proteins Using Cyanogen Bromide ................... 16-16
Alternate Protocol 4: Chemical Cleavage of
Fusion Proteins Using Hydroxlamine ................ 16-16
Alternate Protocol 5: Chemical Cleavage of
Fusion Proteins by Hydrolysis at Low pH ........... 16-17
16.5. Expression and Purification of Glutathione-5-
Transferase Fusion Proteins ....................... 16-18
Basic Protocol: Expression and Purification of
Glutathione-S-Transferase Fusion Proteins ......... 16-18
16.6. Expression and Purification of Thioredoxin
Fusion Proteins ................................... 16-21
Basic Protocol: Construction and Expression of a
Thioredoxin Fusion Protein ........................ 16-21
Support Protocol 1: E. coli Lysis Using a
French Pressure Cell .............................. 16-24
Support Protocol 2: Osmotic Release of
Thioredoxin Fusion Proteins ....................... 16-26
Support Protocol 3: Purification of Thioredoxin
Fusion Proteins by Heat Treatment ................. 16-26
16.7. Overview of the Baculovirus Expression System ..... 16-27
Baculovirus Expression System ..................... 16-27
Posttranslational Modification of Proteins
in Insect Cells ................................... 16-29
Steps for Overproducing Proteins Using the
Baculovirus System ................................ 16-29
Choosing a Baculovirus Transfer Vector ............ 16-30
Choosing a Baculovirus DNA ........................ 16-32
Reagents, Solutions, and Equipment for the
Baculovirus System ................................ 16-33
16.8. Maintenance of Insect Cell Cultures and
Generation of Recombinant Baculoviruses ........... 16-34
Basic Protocol 1: Maintenance and Culture of
Insect Cells ...................................... 16-34
Basic Protocol 2: Cotransfection of Insect
Cells Using Linearized Baculoviral DNA ............ 16-36
Alternate Protocol: Generation of Recombinant
Baculovirus Using Wild-Type Baculoviral DNA ....... 16-38
Basic Protocol 3: Preparation of Baculovirus
Stocks ............................................ 16-41
Basic Protocol 4: Titering Baculovirus Stocks
Using Plaque Assay ................................ 16-42
16.9. Expression and Purification of Recombinant
Proteins Using the Baculovirus System ............. 16-44
Basic Protocol 1: Small-Scale Expression for
Initial Analysis .................................. 16-44
Support Protocol 1: Determining Time Course of
Maximum Protein Production ........................ 16-45
Support Protocol 2: Metabolic Labeling of
Recombinant Proteins .............................. 16-46
Basic Protocol 2: Large-Scale Production of
Recombinant Proteins .............................. 16-47
Basic Protocol 3: Purification of Recombinant
Proteins Containing a Polyhistidine (6xHis)Tag .... 16-48
Alternate Protocol: Purification of Recombinant
Proteins Containing a GST Tag ..................... 16-50
16.10.Overview of Protein Expression in Mammalian
Cells ............................................. 16-51
Viral-Mediated Gene Transfer ...................... 16-51
Transient Expression .............................. 16-52
Stable DNA Transfection ........................... 16-53
Amplification of Transfected DNA .................. 16-54
Expression Vectors ................................ 16-55
Choice of Expression System ....................... 16-55
Troubleshooting ................................... 16-55
16.11.Transient Expression of Proteins Using
COS Cells ......................................... 16-56
Basic Protocol .................................... 16-56
16.12.Overview of the Vaccinia Virus Expression
System ............................................ 16-58
Vaccinia Replication Cycle ........................ 16-59
Effects of Vaccinia Infection ..................... 16-60
Vaccinia Vector Expression System ................. 16-60
Steps for Expression of Genes Using Vaccinia
Vectors ........................................... 16-61
Safety Precautions for Using Vaccinia ............. 16-61
16.13.Preparation of Cell Cultures and Vaccinia
Virus Stocks ...................................... 16-62
Basic Protocol 1: Culture of Monolayer Cells ...... 16-63
Basic Protocol 2: Culture of Cells in
Suspension ........................................ 16-64
Basic Protocol 3: Preparation of a Vaccinia
Virus Stock ....................................... 16-65
Support Protocol 1: Titration of Vaccinia Virus
Stocks by Plaque Assay ............................ 16-66
Basic Protocol 4: Preparation of Chicken Embryo
Fibroblasts ....................................... 16-67
Basic Protocol 5: Preparation of an MVA Stock ..... 16-68
Support Protocol 2: Titration of MVA Stocks by
Immunostaining .................................... 16-69
16.14.Generation of Recombinant Vaccinia Viruses ........ 16-71
Basic Protocol 1: Transfection of Infected Cells
with a Vaccinia Vector ............................ 16-71
Support Protocol 1: Purification of Vaccinia
Virus ............................................. 16-75
Support Protocol 2: Isolation of Vaccinia
Virus DNA ......................................... 16-77
Basic Protocol 2: Selection and Screening of
Recombinant Virus Plaques ......................... 16-78
Basic Protocol 3: Amplification of a Plaque ....... 16-81
Basic Protocol 4: Live Immunostaining of MVA
Recombinants ...................................... 16-82
Support Protocol 3: Coating Plates with
Concanavalin A .................................... 16-83
16.15.Characterization of Recombinant Vaccinia
Viruses and Their Products ........................ 16-84
Basic Protocol 1: Detection of Vaccinia DNA
Using PCR ......................................... 16-84
Basic Protocol 2: Detection of Vaccinia DNA
Using Southern Blot Hybridization ................. 16-87
Basic Protocol 3: Detection of Vaccinia DNA
Using Dot-Blot Hybridization ...................... 16-88
Alternate Protocol: Detection of Expressed
Protein by a Dot-Blot Procedure ................... 16-89
Basic Protocol 4: Detection of Expressed Protein
Using Immunoblotting .............................. 16-90
Basic Protocol 5: Detection of Expressed Protein
Using Immunoprecipitation ......................... 16-91
16.16.Gene Expression Using the Vaccinia Virus/T7 RNA
Polymerase Hybrid System .......................... 16-92
Basic Protocol 1: Liposome-Mediated Transfection
Following Recombinant Vaccinia Virus (vTF7-3)
Infection ......................................... 16-93
Basic Protocol 2: Coinfection with Two
Recombinant Vaccinia Viruses ...................... 16-95
Basic Protocol 3: Infection of OST7-1 Cells
with a Single Virus ............................... 16-96
Basic Protocol 4: Gene Expression Using the
VOTE System ....................................... 16-97
Support Protocol: Detection of Expressed
Protein Using Pulse Labeling ...................... 16-99
16.17.Inducible Gene Expression Using an
Autoregulatory, Tetracycline-ControUed System .... 16-100
Basic Protocol: Calcium Phosphate-Mediated
Stable Transfection of NIH3T3 Cells with pTet-
tTAk and Tetracyline-Regulated Target Plasmids ... 16-102
Support Protocol: Analysis of Target Gene
Protein Expression ............................... 16-105
16.18.Expression and Purification of Epitope-Tagged
Multisubunit Protein Complexes from Mammalian
Cells ............................................ 16-106
Basic Protocol 1: Purification of Multisubunit
Protein Complexes from Clonal Cell Lines
Constitutively Expressing a Flag-Tagged
Protein .......................................... 16-107
Basic Protocol 2: Purification of Multisubunit
Protein Complexes from Clonal Cell Lines
Conditionally Expressing the Flag-Tagged
Protein .......................................... 16-110
Alternate Protocol: Purification of Multiple
Forms of Epitope-Tagged Protein Complexes by
Varying the Starting Material and Wash
Conditions ....................................... 16-113
Support Protocol: Purification of Multiple
Forms of Epitope-Tagged Protein Complexes
Following a P11 Ion-Exchange Chromatographic
Column ........................................... 16-115
17. Analysis of Protein Phosphorylation ...................... 17-1
17.1. Overview of Protein Phosphorylation ................ 17-2
17.2. Labeling Cultured Cells with 32P1 and Preparing
Cell Lysates for Immunoprecipitation ............... 17-4
Basic Protocol: Labeling Cultured Cells with 32P
and Lysis Using Mild Detergent ..................... 17-4
Alternate Protocol: Lysis of Cells by Boiling
in SDS ............................................. 17-6
17.3. Phosphoamino Acid Analysis ......................... 17-7
Basic Protocol: Acid Hydrolysis and Two-
Dimensional Electrophoretic Analysis of
Phosphoamino Acids ................................. 17-7
Alternate Protocol: Alkali Treatment to Enhance
Detection of Tyr- and Thr-Phosphorylated
Proteins Blotted onto Filters ..................... 17-11
17.4. Analysis of Phosphorylation of Unlabeled
Proteins .......................................... 17-12
Basic Protocol 1: Immunoblotting with Anti-
Phosphotyrosine Antibodies and Detection Using
[125I]Protein A ................................... 17-12
Alternate Protocol: Detection of Bound
Antibodies by Enhanced Chemiluminescence (ECL) .... 17-13
Basic Protocol 2: Identification of
Phosphorylated Proteins by Phosphatase
Digestion ......................................... 17-14
17.5. Detection of Phosphorylation by Enzymatic
Techniques ........................................ 17-15
Basic Protocol 1: Digestion of Phosphoproteins
with Nonspecific Acid Phosphatases ................ 17-16
Alternate Protocol 1: Digestion of
Phosphoproteins with Nonspecific Alkaline
Phosphatase ....................................... 17-16
Basic Protocol 2: Digestion of Phosphoproteins
with Protein Serine/Threonine Phosphatases ........ 17-17
Alternate Protocol 2: Digestion of
Phosphoproteins with Protein Tyrosine
Phosphatases ...................................... 17-18
Support Protocol: Measurement and Identification
of Released 32P ................................... 17-18
17.6. Production of Antibodies that Recognize Specific
Tyrosine-Phosphorylated Peptides .................. 17-19
Basic Protocol 1: Production of Polyclonal
Anti-Phosphopeptide Antibodies .................... 17-19
Basic Protocol 2: Production of Monoclonal
Anti-Phosphopeptide Antibodies .................... 17-21
Support Protocol 1: Synthesis of Peptides ......... 17-24
Support Protocol 2: Coupling of Peptides to
Affi-Gel 10 Affinity Matrix ....................... 17-24
Support Protocol 3: Coupling of Phosphotyrosine
to Affi-Gel 10 Affinity Matrix .................... 17-25
17.7. Assays of Protein Kinases Using Exogenous
Substrates ........................................ 17-26
Strategic Planning ................................ 17-26
Basic Protocol 1: Assay for Cyclic Nucleotide-
Dependent Protein Kinases ......................... 17-29
Basic Protocol 2: Assay for Protein Kinase C
Isoforms .......................................... 17-30
Basic Protocol 3: Assay for Casein Kinases
Using β-Casein .................................... 17-31
Alternate Protocol: Assay for Casein Kinases
Using a Peptide Substrate ......................... 17-31
Basic Protocol 4: Assay for Ca2+/Calmodulin-
Dependent Kinases ................................. 17-32
Basic Protocol 5: Assay for Tyrosine Kinases ...... 17-33
Basic Protocol 6: In-Gel Protein Kinase Assays .... 17-34
Support Protocol 1: Preparing a Cell Lysate for
Kinase Assays ..................................... 17-35
Support Protocol 2: TCA Precipitation to
Determine Incorporation of Radioactivity .......... 17-36
Support Protocol 3: Adsorption onto P81
Phosphocellulose Paper ............................ 17-37
17.8. Permeabilization Strategies to Study Protein
Phosphorylation ................................... 17-39
Basic Protocol: Analysis of Protein
Phosphorylation in Permeabilized Cells ............ 17-39
Intact Cell Sample Preparation for
Electrophoretic Analysis of Protein
Phosphorylation ................................... 17-42
Alternate Protocol 1: Intact Cell Sample
Preparation for SDS-PAGE .......................... 17-42
Alternate Protocol 2: Intact Cell Sample
Preparation for Isoelectric Focusing .............. 17-43
17.9. Phosphopeptide Mapping and Identification of
Phosphorylation Sites ............................. 17-43
Basic Protocol 1: Tryptic Phosphopeptide Mapping
of Proteins Isolated from SDS-Polyacrylamide
Gels .............................................. 17-44
Alternate Protocol: Proteolytic Digestion of
Immobilized Proteins .............................. 17-50
Support Protocol 1: Isolation of Phosphopeptides
from the Cellulose Plate .......................... 17-51
Basic Protocol 2: Determination of the Position
of the Phosphorylated Amino Acid in the Peptide
by Manual Edman Degradation ....................... 17-53
Basic Protocol 3: Diagnostic Secondary Digests
to Test for the Presence of Specific Amino Acids
in the Phosphopeptide ............................. 17-55
Support Protocol 2: Preparation of
Phosphopeptides for Microsequence Determination
or Mass Spectrometry .............................. 17-57
18. Informatics for Molecular Biologists ..................... 18-1
18.1. Sequence Similarity Searching Using the BLAST
Family of Programs ................................. 18-1
Accessing BLAST Programs and Documentation ......... 18-2
Introduction to BLAST .............................. 18-3
Examples of BLAST Searches ......................... 18-6
Searching Strategies .............................. 18-19
Appendix A: BLAST Parameters ...................... 18-23
Appendix B: Sequence Identifier Syntax ............ 18-23
19. Analysis of Protein Interactions ......................... 19-1
Introduction ............................................. 19-1
Equilibrium Parameters ................................... 19-1
What This Chapter Describes .............................. 19-4
19.1. Interaction Trap/Two-Hybrid System to Identify
Interacting Proteins ............................... 19-5
Basic Protocol 1: Characterizing a Bait Protein .... 19-7
Basic Protocol 2: Performing an Interactor Hunt ... 19-13
Alternate Protocol 1: Rapid Screen for
Interaction Trap Positives ........................ 19-21
Alternate Protocol 2: Performing a Hunt by
Interaction Mating ................................ 19-22
Support Protocol 1: Preparation of Protein
Extracts for Immunoblot Analysis .................. 19-25
Support Protocol 2: Preparation of Sheared
Salmon Sperm Carrier DNA .......................... 19-25
19.2. Affinity Purification of Proteins Binding to GST
Fusion Proteins ................................... 19-27
Basic Protocol: GST Fusion Protein-Affinity
Purification ...................................... 19-27
Support Protocol: Preparation of E. coli
Extract ........................................... 19-30
19.3. Phage-Based Expression Cloning to Identify
Interacting Proteins .............................. 19-31
Strategic Planning ................................ 19-32
Basic Protocol: Interaction Cloning ............... 19-32
19.4. Surface Plasmon Resonance for Measurements of
Biological Interest ............................... 19-35
Basic Protocol 1: SPR Using Biacore Chips ......... 19-35
Basic Protocol 2: SPR Using NTA-SAM Chips ......... 19-37
19.5. Detection of Protein-Protein Interactions
by Coprecipitation ................................ 19-38
Basic Protocol: Coprecipitating Proteins with
Protein A/G-Sepharose ............................. 19-38
Alternate Protocol: Coprecipitating a GST
Fusion Protein .................................... 19-39
19.6. Identification of Protein Interactions by Far
Western Analysis .................................. 19-40
Basic Protocol: Far Western Analysis of a
Protein Mixture ................................... 19-40
Alternate Protocol 1: Detecting Interacting
Proteins by Immunoblotting ........................ 19-41
Alternate Protocol 2: Using Peptides to
Identify Specific Interacting Sequences
in a Far Western Blot ............................. 19-43
20. Chromatin Assembly and Analysis .......................... 20-1
20.1. Micrococcal Nuclease Analysis of Chromatin
Structure .......................................... 20-2
Basic Protocol 1: Micrococcal Nuclease Digestion
of Chromatin in Permeabilized Cells ................ 20-2
Basic Protocol 2: Micrococcal Nuclease Digestion
of Chromatin in Isolated Nuclei .................... 20-3
Basic Protocol 3: Micrococcal Nuclease Digestion
of Purified Genomic DNA ............................ 20-5
Support Protocol 1: Purification and
Characterization of DNA from Chromatin
Digestions ......................................... 20-6
Support Protocol 2: Nuclease Cleavage Mapping
Strategies ......................................... 20-8
Support Protocol 3: Using a Modified LM-PCR
Procedure to Map Double-Stranded MNase Cleavages
at the Nucleotide Level of Resolution ............. 20-10
20.2. Separation of Histone Variants and Post-
Translationally Modified Isoforms by
Triton/Acetic Acid/Urea Polyacrylamide
Gel Electrophoresis ............................... 20-11
Basic Protocol: Triton/Acetic Acid/Urea (TAU)
Polyacrylamide Gel Electrophoresis for Analysis
of Histones ....................................... 20-11
Support Protocol 1: Assembly of Gel Plates ........ 20-14
Support Protocol 2: Histone Isolation from
Prepared Nuclei ................................... 20-14
Support Protocol 3: Electrophoretic Transfer of
TAU-Polyacrylamide Gels ........................... 20-15
20.3. Characterization of Proteins Bound to Chromatin
by Immunoprecipitation from Whole-Cell Extracts ... 20-16
Basic Protocol: Characterization of Proteins
Bound to Chromatin by Immunoprecipitation from
Whole-Cell Extracts ............................... 20-16
20.4. Isolation of Histones and Nucleosome Cores from
Mammalian Cells ................................... 20-20
Basic Protocol 1: Preparation of a Washed
Nuclear Pellet .................................... 20-20
Basic Protocol 2: Solubilization and
Purification of Histone HI-Depleted
Oligonucleosomes .................................. 20-21
Basic Protocol 3: Purification of Mono- and
Dinucleosomes ..................................... 20-23
Basic Protocol 4: Purification of Core Histones
by Hydroxylapatite Chromatography ................. 20-24
20.5. Assembly of Nucleosomal Templates by Salt
Dialysis .......................................... 20-25
Basic Protocol 1: Assembly of Nucleosomal
Templates by Step Salt Dialysis ................... 20-26
Basic Protocol 2: Assembly of High
Concentration of Mononucleosomes by Gradient
Salt Dialysis ..................................... 20-27
Basic Protocol 3: Assembly of Nucleosomal
Arrays by Gradient Salt Dialysis .................. 20-28
Support Protocol 1: Purification of Recombinant
Core Histones from Bacteria ....................... 20-29
Support Protocol 2: Preparation of Single 5'
End-Labeled DNA for Mononucleosome Assembly ....... 20-30
Support Protocol 3: Preparation of DNA for
Assembly of High Concentration Unlabeled
Mononucleosomes ................................... 20-31
Support Protocol 4: Preparation of DNA for
Nucleosomal Arrays ................................ 20-32
Support Protocol 5: Analysis of Reconstituted
Complexes by Agarose Gel Electrophoresis .......... 20-32
Support Protocol 6: Analysis of Reconstituted
Complexes by Polyacrylamide Gel Electrophoresis ... 20-33
Support Protocol 7: EcoRI Digestion to
Determine Extent of G5E4 Array Assembly ........... 20-34
20.6. Chromatin Assembly Using Drosophila Systems ....... 20-34
Basic Protocol 1: Preparation of the Drosophila
S-190 Chromatin Assembly Extract .................. 20-34
Basic Protocol 2: Purification of Core
Histones from the Drosophila Embryos .............. 20-36
Basic Protocol 3: Chromatin Assembly with the
S-190 Extract ..................................... 20-38
Basic Protocol 4: Expression and Purification
of the Recombinant Drosophila Acf ................. 20-40
Basic Protocol 5: Expression and Purification
of the Recombinant Drosophila NAP-1 ............... 20-41
Alternate Protocol 1: Expression and
Purification of the Recombinant Drosophila
NAP-1 (NTA Superflow Resin) ....................... 20-43
Basic Protocol 6: Chromatin Assembly with
Purified Recombinant Drosophila Factors ........... 20-43
Alternate Protocol 2: Titration of the Ratio
of Core Histones to DNA in the Recombinant
Chromatin Assembly Reaction ....................... 20-45
Support Protocol: Expression and Purification
of the Core Catalytic Domain of the Drosophila
Topoisomerase I ................................... 20-46
21. Nucleic Acid Arrays ...................................... 21-1
21.1. Overview of Nucleic Acid Arrays .................... 21-1
What Are Microarrays Good For? ..................... 21-2
What Else Are Nucleic Acid Microarrays
Good For? .......................................... 21-3
What About Data Analysis? .......................... 21-5
Where Can I Get More Information? .................. 21-5
21.2. Preparation of mRNA for Expression Monitoring ...... 21-6
Strategic Planning ................................. 21-6
Basic Protocol: Amplification of mRNA for
Expression Monitoring and Hybridization to
Oligonucleotide Array Chips ........................ 21-7
Support Protocol 1: In Vitro Transcription of
Control Genes and Preparation of Transcript
Pools ............................................. 21-12
Alternate Protocol: Solid-Phase Reversible
Immobilization (SPRI) Purification of cDNA
and IVT Products .................................. 21-14
Support Protocol 2: Quantitation of cDNA .......... 21-15
21.3. Profiling Human Gene Expression with cDNA
Microarrays ....................................... 21-16
Basic Protocol 1: cDNA Amplification and
Printing .......................................... 21-16
Basic Protocol 2: RNA Extraction and Labeling ..... 21-21
Basic Protocol 3: Hybridization and Data
Extraction ........................................ 21-24
Support Protocol 1: Agarose Gel Electrophoresis
of ESTs ........................................... 21-26
Support Protocol 2: Fluorometric Determination
of DNA Concentration .............................. 21-28
Support Protocol 3: Coating Slides with
Poly-L-Lysine ..................................... 21-29
22. Generation and Use of Combinatorial Libraries ............ 22-1
22.1. Design, Synthesis, and Amplification of DNA
Pools for Construction of Combinatorial Pools
and Libraries ...................................... 22-1
Basic Protocol 1: Purification of a Random
Sequence Pool ...................................... 22-1
Support Protocol 1: Determining the Pool
Complexity ......................................... 22-3
Support Protocol 2: Determining the Pool Bias ...... 22-4
Support Protocol 3: Small-Scale PCR Optimization
of Pool Amplification .............................. 22-4
Basic Protocol 2: Large-Scale PCR Amplification
of Pool DNA ........................................ 22-5
22.2. Peptide Aptamers: Dominant "Genetic" Agents
for Forward and Reverse Analysis of Cellular
Processes .......................................... 22-7
Basic Protocol 1: Construction of a Combinatorial
Thioredoxin Peptide Aptamer Library ................ 22-8
Basic Protocol 2: Isolation of Peptide Aptamers
for Specific Proteins Using the Interaction Trap
Two-Hybrid System ................................. 22-10
Basic Protocol 3: Defining Recognition
Specificity with Interaction Mating ............... 22-12
Basic Protocol 4: Affinity Maturation of
Peptide Aptamers .................................. 22-13
Basic Protocol 5: Forward Analysis of Cellular
Processes Using Peptide Aptamers .................. 22-15
Support Protocol: Identification of Peptide
Aptamer Targets ................................... 22-16
22.3. Protein Selection Using mRNA Display .............. 22-17
Basic Protocol 1: Preparation and Purification
of mRNA-Displayed Proteins ........................ 22-17
Basic Protocol 2: Purification and Reverse
Transcription of the mRNA-Displayed Proteins ...... 22-20
Basic Protocol 3: Selection and Amplification of
the mRNA-Displayed Proteins ....................... 22-23
Support Protocol 1: Flag Tag Purification ......... 22-27
Support Protocol 2: Mutagenic PCR ................. 22-28
23. Discovery and Analysis of Differentially Expressed
Genes in Single Cells and Cell Populations ............... 23-1
23.1. Production of a Subtracted cDNA Library ............ 23-2
Basic Protocol: Production of a Subtracted Library ........... 23-2
23.2. PCR-Based Subtractive cDNA Cloning ................. 23-6
Basic Protocol: Construction of Subtracted cDNA
Libraries .......................................... 23-6
Support Protocol: Slot Blot Hybridization to
Monitor Subtraction ............................... 23-12
23.3. Differential Display of mRNA by PCR ............... 23-14
Basic Protocol: Differential Display of mRNA
by PCR ............................................ 23-14
23.4. Restriction-Mediated Differential Display
(RMDD) ............................................ 23-18
Strategic Planning ................................ 23-18
Basic Protocol: RMDD Library Preparation and
Two-Round Amplification ........................... 23-19
Alternate Protocol: Amplification by Two-Phase
PCR ............................................... 23-24
Support Protocol: Direct Blotting
Electrophoresis ................................... 23-25
23.5. AFLP-Based Transcript Profiling ................... 23-27
Basic Protocol: AFLP-Based Transcript Profiling ... 23-27
23.6. Serial Analysis of Gene Expression (SAGE) ......... 23-34
Basic Protocol: Serial Analysis of Gene
Expression (SAGE) ................................. 23-34
Support Protocol 1: Verifying cDNA Production
by PCR Analysis ................................... 23-46
Support Protocol 2: Optimizing Ditag PCR
Amplification ..................................... 23-46
Appendices
Al Reagents and Solutions .............................. Al-1
A2 Useful Measurements and Data ........................ A2-1
A3 Commonly Used Techniques in Biochemistry
and Molecular Biology ............................... A3-1
3A Detection and Quantitation of Radiolabeled
Proteins and DNA in Gels and Blots .................. A3-1
3B Silanizing Glassware ............................. A3-9
3C Dialysis and Ultrafiltration .................... A3-10
3D Quantitation of DNA and RNA with Absorption
and Fluorescence Spectroscopy ................... A3-15
3E Introduction of Restriction Enzyme
Recognition Sequences by Silent Mutation ........ A3-20
3F Techniques for Mammalian Cell Tissue Culture .... A3-22
3G Safe Use of Radioisotopes ....................... A3-31
3H Statistics for the Molecular Biologist:
Group Comparisons ............................... A3-43
A4 Selected Suppliers of Reagents and Equipment ........ A4-1
References .......................................... 1
Index
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