Patch Clamp Methods and Protocols surveys the typical patch clamp applications and advises scientists on identifying problems and selecting the best technique in each instance. The experiments described require a basic level of electrophysiological training and aid the researcher in pursuing new areas of electrophysiology and using the patch clamp technique effectively. Patch Clamp Methods and Protocols is divided into three sections that cover the major areas of patch clamp application: Pharmacology, Physiology, and Biophysics. The first section provides examples and step by step instructions on how to use whole-cell and single-channel patch clamp methods for testing drugs in industrial settings. The second section provides a wide selection of patch clamp applications in physiological studies. The last part focuses on the biophysical applications of the patch clamp method using single channel recordings or statistical analysis of whole-cell currents in order to obtain parameters that describe ion channel properties or transmitter release. Individual techniques are explored within the area that they are applied most often. Researchers will find Patch Clamp Methods and Protocols to be an invaluable aid in the design and execution of a wide variety of patch clamp experiments, both on their own and in conjunction with other state-of-the-art methodologies.

Patch Clamp Methods and Protocols surveys the typical patch clamp applications and advises scientists on identifying problems and selecting the best technique in each instance. The experiments described require a basic level of electrophysiological training and aid the researcher in pursuing new areas of electrophysiology and using the patch clamp technique effectively. Patch Clamp Methods and Protocols is divided into three sections that cover the major areas of patch clamp application: Pharmacology, Physiology, and Biophysics. The first section provides examples and step by step instructions on how to use whole-cell and single-channel patch clamp methods for testing drugs in industrial settings. The second section provides a wide selection of patch clamp applications in physiological studies. The last part focuses on the biophysical applications of the patch clamp method using single channel recordings or statistical analysis of whole-cell currents in order to obtain parameters that describe ion channel properties or transmitter release. Individual techniques are explored within the area that they are applied most often. Researchers will find Patch Clamp Methods and Protocols to be an invaluable aid in the design and execution of a wide variety of patch clamp experiments, both on their own and in conjunction with other state-of-the-art methodologies.

Pharmacological analysis of recombinant NR1a/2A and NR1a/2B NMDA receptors using the whole-cell patch-clamp method / László Fodor and József Nagy Memantine as an example of a fast, voltage-dependent, open channel N-methyl-D-aspartate receptor blocker / Chris G. Parsons and Kate Gilling Methods for evaluation of positive allosteric modulators of glutamate AMPA receptors / Edward R. Siuda, Jennifer C. Quirk, and Eric S.Nisenbaum Automated voltage-clamp technique / Andrea Ghetti, António Guia, and Jia Xu Flip-the-tip : automated patch clamping based on glass electrodes / Michael Fejtl ... [et al.] The roboocyte : automated electrophysiology based on Xenopus oocytes / Christine Leisgen, Mike Kuester, and Christoph Methfessel Infrared-guided laser stimulation as a tool for elucidating the synaptic site of expression of long-term synaptic plasticity / Gerhard Rammes ... [et al.] Single-cell RT-PCR, a technique to decipher the electrical, anatomical, and genetic determinants of neuronal diversity / Maria Toledo-Rodriguez and Henry Markram Mechanosensitive ion channels investigated simultaneously by scanning probe microscopy and patch clamp / Matthias G. Langer Synaptic connectivity in engineered neuronal networks / Peter Molnar ... [et al.] Modeling of action potential generation in NG108-15 cells / Peter Molnar and James J. Hickman Whole-cell voltage clamp on skeletal muscle fibers with the silicone-clamp technique / Sandrine Pouvreau ... [et al.] Determination of channel properties at the unitary level in adult mammalian isolated cardiomyocytes / Romain Guinamard Electrophysiological properties of embryonic stem cells during differentiation into cardiomyocyte-like cell types / Antoni C.G. van Ginneken and Arnoud C. Fijnvandraat Hybrid neuronal network studies under dynamic clamp / Alan D. Dorval II ... [et al.] Cardiac channelopathies studied with the dynamic action potential-clamp technique / Géza Berecki ... [et al.] Principles of single-channel kinetic analysis / Feng Qin Use of Xenopus oocytes to measure ionic selectivity of pore-forming peptides and ion channels / Thierry Cens and Pierre Charnet Estimation of quantal parameters with multiple-probability fluctuation analysis / Chiara Saviane and Robin Angus Silver.

This volume describes a range of standard and novel methodological approaches used to probe ion channel function across different modalities. Chapters guide readers through methods and protocols from an introduction to the decades old patch clamp method for the ion channel neophyte to more complex, recent protocol advances, such as optogenetics. Written in the highly successful __Methods in Molecular Biology__ series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, __Patch Clamp Electrophysiology: Methods and Protocols__ aims to be a reference guide for current and future ion channel physiologists.

2-nd ed. - Humana Press, Totowa NJ.- 2007.- 608 p. This second edition of Electron Microscopy: Methods and Protocols is written for established researchers as well as new students in the field of molecular biology. It is not only for biomedical but also for general biological science research and its application. The combination of microscopical and chemical analyses has provided the basis for our current understanding of cell biology. The high-resolution electron microscope with its associated equipment can serve as a powerful tool for analyzing molecular structure, interactions, and processes. The second edition now consists of two main areas: the first relates to TEM, and the second covers scanning electron microscopy (SEM) and mass spectrometry (MS). The TEM area comprises several sections: Conventional and microwave-assisted specimen preparation methods for cultured cells and biomedical and plant tissues, for the benefit of those who only require routine TEM images through ultramicrotomy sectioning and then positive staining. Cryo-specimen preparation by high-pressure freezing and cryoultramicrotomy. Negative staining and immunogold labeling techniques for samples prepared through conventional, cryosectioning, or high-pressure freezing methods. Some of these chapters are presented in correlative approaches using TEM with fluorescent or confocal microscopy. Quantitative aspects of immunogold labeling in resin embedded samples are also included. TEM crystallography and cryo-TEM tomography for the study of membrane proteins, macromolecules, organelles, and cells. One chapter on the application of EELS to biomedical tissue is included.

In the dramatic and rapidly developing field of neural transplantation for CNS repair, the most powerful contributor has been the vital research focusing on stem cells. In __Neural Cell Transplantation: Methods and Protocols__, leading experts in the field examine tried-and-true laboratory techniques in order to supply scientists with a firm foundation upon which further advancements can be based. Written in the highly successful __Methods in Molecular BiologyTM__ series format, the chapters of this volume include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and Notes sections, which examine tips on troubleshooting and avoiding known pitfalls. Cutting-edge and easy to use, __Neural Cell Transplantation: Methods and Protocols__ provides the most thorough and essential protocols that will allow new generations of neuroscientists to enter and contribute to this uniquely inspiring field.

Second Edition - Humana Press Inc., Totowa, NJ, 2003.- 457 p. Antibody Preparation Tissue Preparation fori Light Microscopic Analyses Light Microscopic Detection Systems Fluorescence-Activated Cell Sorter (FACS] Analyses Colloidal Gold Detection Systems ton Electron Microscopic Analyses Application for Acid Localization by in situ Hybridization Special Applications for the Clinical Laboratory

This second edition provides detailed techniques used for the study and characterization of the plant vascular system, with a central focus on the xylem tissue. Chapters are organized in three main sections covering; analysis of xylem development, xylem characterization though imaging techniques, and analysis of the xylem composition. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Xylem: Methods and Protocols, Second Edition aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

CH1. Flow Cytometry. An introduction......Page 2 CH2. Multiparameter Flow Cytometry of Bacteria......Page 34 CH3. Multiparameter Data Acquisition and Analysis of Leukocytes......Page 46 CH4. Flow Cytometric Analysis of Kinase Signaling Cascades......Page 68 CH5. Cytokine Flow Cytometry......Page 96 CH6.......Page 110 CH7.......Page 126 CH8. Apoptosis......Page 142 CH9. Detection and Enrichment of Hematopoietic Stem Cells by Side Population Phenotype......Page 162 CH10......Page 182 CH11. Phenotypic and Functional Analyses of CD34neg Hematopoietic Precursos From Mobilized Peripheral Blood......Page 202 CH12. Multiparameter Flow Cytometry of Fluorescent Protein Reporters......Page 220 CH13. Analysis of Fluorescent Protein Expressing Cells by Flow Cytometry......Page 240 CH14.......Page 260 CH15.......Page 282 CH16.......Page 294 CH17.......Page 312 CH18.......Page 334 CH19......Page 346 CH20......Page 356 CH21......Page 372 CH22. Telomere Length Measurement by Fluorescent In Situ Hybridization and Flow Cytometry......Page 386 CH23.......Page 400 CH24.......Page 420

This second edition provides up-to-date protocols focusing on techniques for the specific enrichment of phosphopeptides and phosphoproteins. __Phosphoproteomics Methods and Protocols, Second Edition__ guides readers through different labeling strategies for quantitative phosphoproteomics; high-throughput mass spectrometry-based phosphoproteome analyses and phospho flow cytometry; and bioinformatics strategies for phosphoproteomics data analysis and integration. Additional protocols concentrate on the identification of kinase-substrate relationships by both high- and low-throughput approaches. Written in the highly successful __Methods in Molecular Biology__ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, __Phosphoproteomics Methods and Protocols, Second Edition__ aims to ensure successful results in the further study of this vital field.

In __Mouse Molecular Embryology: Methods and Protocols__, expert researchers in the field detail many of the protocols used to study mouse embryology. These include protocols and techniques that are "close to the embryo": such as, manipulating embryonic gene expression, culturing explanted embryonic tissue and harvesting embryonic RNA. With additional chapters on fluorescence imaging, lineage tracing, and genetic ablation. Written in the highly successful __Methods in Molecular Biology__ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, __Mouse Molecular Embryology: Methods and Protocols__ seeks to aid scientist in the further study of mouse embryo and its relation to other aspects of biological research.

Human Lens Epithelial Cell Culture......Page 2 Human Airway Epithelial Cell Culture......Page 8 Rat Gastric Mucosal Epithelial Cell Culture......Page 18 Thymic Epithelial Cell Culture......Page 27 Bile Duct Epithelial Cell Culture......Page 37 Liver Epithelial Cell Culture......Page 53 Pulmonary Epithelial Cell Culture......Page 64 Prostate Epithelial Cell Isolation and Culture......Page 75 A Bovine Mammary Endothelial/Epithelial Cell Culture Model of the Blood/Milk Barrier......Page 83 The Blood–CSF Barrier in Culture......Page 97 An In Vitro Model of Differentiated Human Airway Epithelia......Page 113 Primary Mouse Keratinocyte Culture......Page 136 Analyzing Apoptosis in Cultured Epithelial Cells......Page 142 Keratins as Markers of Epithelial Cells......Page 153 Epithelial Cell Integrins......Page 164 Isolation, Cultivation, and Differentiation of Normal Human Epidermal Keratinocytes in Serum-Free Medium......Page 173 Clinical Application of Autologous Cultured Epithelia for the Treatment of Burns and Disfigurement of Skin Surfaces......Page 179 Transplantation of Cultured Epithelial Cells......Page 191 An Outgrowth Culture System of Normal Human Epithelium......Page 206 Application of Epithelial Cell Culture in Drug Transport in the Respiratory Tract......Page 210 Applications of Epithelial Cell Culture in Studies of Drug Transport......Page 226 X-Ray Microanalysis of Epithelial Cells in Culture......Page 266 ENU Mutagenesis of Rat Mammary Epithelial Cells......Page 283 ENU-Induced Ovarian Cancer......Page 296 Measurement of Membrane Capacitance in Epithelial Monolayers......Page 306 Assessing Epithelial Cell Confluence by Spectroscopy......Page 319 Co-Cultivation of Liver Epithelial Cells with Hepatocytes......Page 327 Co-Culture and Crosstalk between Endothelial Cells and Vascular Smooth Muscle Cells Mediated by Intracellular Calcium......Page 337 Establishing Epithelial–Immune Cell Co-Cultures......Page 348 Explant Cultures of Embryonic Epithelium......Page 361 Bacterial Interactions with Host Epithelium In Vitro......Page 371

University of Michigan, Ann Arbor. Methods in Molecular Biology, Volume 12. Reference of methods in PFGE - pulsed-field gel electrophoresis, for researchers in molecular biology, biology, or physics. Includes specific methods: CHEF, OFAGE, ROFE, ODPFGE, others.

This volume detials diverse methodological approaches on the assembly and applications of DNA origami assemblies. Chapters guide readers through different synthetic and computational methods, isolation and structural characterization of 2D and 3D DNA origami nanoarchitectures, nanophotonics, drug delivery, biophysics, and synthetic biology.Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, DNA and RNA Origami: Methods and Protocols aims to serve as a guideline describing the current state-of-the-art assembly methodologies and applications of DNA origami nanostructures.

This volume presents protocols on yeast cytokinesis that will help Molecular and Cellular Biology researchers in the use of these microorganisms to approach the study of general or specific key questions in cytokinesis. Written for the __Methods in Molecular Biology__ series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, __Yeast Cytokinesis: Methods and Protocols__ provides practical and step-by-step detailed protocols useful for a wide audience ranging from experienced researchers to beginners in the use of yeasts.

1 - The Simultaneous Isolationof RNA and DNAfrom Tissues and Cultured Cells......Page 1 2 - Restriction Endonuclease Digestionof DNA......Page 9 3 - Agarose Gel Electrophoresis......Page 15 4 - Capillary Blotting of Agarose Gels......Page 20 5 - Random Primed 32P-Labeling of DNA......Page 25 6 - Hybridization and CompetitionHybridization of Southern Blots......Page 29 7 - Utilization of DNA Probeswith Digoxigenin-Modified Nucleotidesin Southern Hybridizations......Page 39 8 - The Preparation of Fluorescein-LabeledNucleic Acid Probes and Their DetectionUsing Alkaline Phosphatase-CatalyzedDioxetane Chernilurninescence......Page 51 9 - Monitoring Incorporation of Fluoresceininto Nucleic Acid ProbesUsing a Rapid Labeling Assay......Page 63 10 - The Preparation of HorseradishPeroxidase-Labeled Nucleic Acid Probesand Their Detection Using EnhancedChemiluminescence......Page 65

1.1. How Much Do I Need?......Page 2 1.3. Do I Need a Completely Pure Protein?......Page 3 1.4. What Source Should I Use?......Page 5 2.1. Solubility......Page 6 2.2. Charge......Page 7 2.4. Specific Binding......Page 8 3. Documenting the Purification......Page 10 4. An Example......Page 11 1. Introduction......Page 16 2. Materials......Page 17 4. Notes......Page 18 1. Introduction......Page 22 2. Materials......Page 23 3.2. Extraction of Cereal Seed Proteins, Using a Modified Osborne Procedure......Page 24 3.3. Extraction of Proteins for SDS-PAGE Analysis......Page 25 4. Notes......Page 26 1. Introduction......Page 30 3.1. Lysis of Escherichia coli and Harvesting of Inclusion Bodies......Page 32 3.4. Refolding of Protein......Page 33 4. Notes......Page 34 1. Introduction......Page 38 3. Methods......Page 39 3.1. Extraction of Intracellular Proteins From Metarhiziumanisopliae......Page 40 3.2. Screening for Extracellular Enzymes......Page 41 4. Notes......Page 42 1. Introduction......Page 48 3.2. Subcellular Fractionation......Page 50 4. Notes......Page 51 1. Introduction......Page 54 2.2. Barley Protoplast and Chloroplast Preparation......Page 55 3.1.1. Wheat Protoplast Preparation......Page 56 3.1.4. Barley Chloroplast Preparation......Page 57 3.3. Assay of Plastid Intactness......Page 58 3.4. Assay of Photosynthetic Activity (CO2-Dependent Oxygen Evolution)of Chloroplasts......Page 59 3.6. Isolation of Mitochondria From Pea Leaves......Page 60 4. Notes......Page 62 1.1. The Problems......Page 66 1.2. Some Solutions......Page 68 2. Materials......Page 69 3.2. Ammonium Sulfate Fractionation of the Extract......Page 70 4. Notes......Page 71 5. Envoi......Page 74 1. Introduction......Page 76 1.1. Recognition of Proteolytic Attack......Page 77 1.2. Strategies for Prevention of Proteolysis......Page 78 1.3. Proteinases and Their Inhibition......Page 79 1.4. Assay of Endopeptidase Activity......Page 81 3.1. Method 1: Working Inhibitor Cocktails......Page 82 3.4. Method 4: Zymography......Page 83 4. Notes......Page 84 1. Introduction......Page 86 3.1. Precipitation With Ammonium Sulfate......Page 87 3.2. Forced Dialysis......Page 88 4. Notes......Page 89 1. Introduction......Page 92 2.1. Preparation of Buffers......Page 94 2.2. Dialysis......Page 95 3.2. Changing Buffers by Dialysis......Page 96 3.3. Changing Buffers by Gel Filtration......Page 97 4. Notes......Page 98 1.1. Membrane Processes......Page 102 1.2. UF Materials and Devices......Page 103 1.3. Operating Parameters......Page 109 1.4. Applications......Page 111 2. Materials......Page 113 3.1. Using a Stirred Cell for Concentration......Page 114 3.2. Protein and Peptide Recovery From Polyacrylamide Gels......Page 115 4. Notes......Page 116 1. Introduction......Page 118 2.2. Fractional Precipitation With Acetone......Page 119 3.2. Fractional Precipitation With Acetone......Page 121 4. Notes......Page 122 1. Introduction......Page 126 2. Materials......Page 128 3.2. Chromatography......Page 129 4. Notes......Page 130 1. Introduction......Page 134 2. Materials......Page 135 4. Notes......Page 136 1.1. General Principles......Page 140 1.2. Affinity Matrices......Page 142 1.3. Practical Aspects......Page 143 2. Materials......Page 146 3. Methods......Page 147 4. Notes......Page 148 1. Introduction......Page 152 2. Materials......Page 153 3.1. Screening Dye Ligands......Page 154 3.3. Example Purification: Isolation of Coenzyme A Synthase......Page 155 4. Notes......Page 156 1. Introduction......Page 160 2.2. Immobilization of Con A on Affigel 15......Page 162 3.3. Purification of Glycoproteins on Immobilized Con A......Page 163 4. Notes......Page 164 1.1. General Principles......Page 168 1.2. Practical Aspects......Page 169 1.3. Practical Aspects......Page 172 2.2. Immunopurification......Page 174 3.3. Immunoaffinity Purification of Target Protein From Crude Extract......Page 175 4. Notes......Page 176 1. Introduction......Page 180 2. Materials......Page 182 2.1. Stationary Phase for IMAC......Page 183 2.3. Buffers......Page 184 3.2. Elution of Adsorbed Proteins......Page 185 3.5. High-Performance IMAC......Page 186 4. Notes......Page 188 1. Introduction......Page 192 4. Notes......Page 193 1. Introduction......Page 196 2.2. Commercially Available Matrices......Page 198 3.1. Thiophilic Chromatography......Page 200 3.3. Hydrophobic Charge Induction Chromatography......Page 202 4. Notes......Page 203 1.1. Overview......Page 206 1.3. Structural and Kinetic Requirements for Affinity PrecipitationWith Bis-Coenzymes......Page 207 2.1. Synthesis of Coenzyme Derivatives......Page 210 2.3. Enzyme Purification......Page 213 3.1. Synthesis of Coenzyme Derivatives......Page 214 3.3. Enzyme Purification Protocols......Page 217 4. Notes......Page 220 References......Page 222 1. Introduction......Page 226 3.1. Gel Preparation......Page 227 3.2. Sample Application......Page 229 3.6. Collection of the Zones......Page 230 3.7. Separation of the Protein From the Carrier Ampholytes......Page 231 4. Notes......Page 232 References......Page 233 2.1. Buffers......Page 234 4. Notes......Page 235 References......Page 238 1. Introduction......Page 240 2.1. Equipment......Page 242 2.3. Selection of Matrix......Page 245 3.2. Preparation and Packing......Page 247 3.5. Evaluation of Column Packing......Page 248 3.6. Standards and Calibration......Page 249 3.8. Column Cleaning and Storage......Page 250 4. Notes......Page 251 References......Page 252 2. Materials......Page 254 3.3. Scouting FPLC Methods......Page 256 4. Notes......Page 257 References......Page 259 1. Introduction......Page 260 1.1. Principle of Separation......Page 261 1.2. RP-HPLC Columns......Page 262 1.3. Mobile Phase......Page 267 1.4. Temperature......Page 272 2.1. Equipment......Page 273 2.5. Detector......Page 274 2.6. Protein Sample......Page 276 4. Notes......Page 277 References......Page 281 1.1. Peripheral Membrane Proteins......Page 284 1.2. Integral Membrane Proteins......Page 285 1.3. Experimental Design......Page 288 2.4. Extraction of the Intrinsic Enzyme Ca2-ATPaseFrom Sarcoplasmic Reticulum......Page 289 3.2. Extraction of the Extrinsic Calcium-Binding Protein CalsequestrinFrom Sarcoplasmic Reticulum......Page 290 3.5. Assay of Ca2-ATPase Activity......Page 291 4. Notes......Page 292 References......Page 293 1. Introduction......Page 296 3. Methods......Page 299 4. Notes......Page 300 References......Page 301 1. Introduction......Page 302 2.1. Isolation of Crude Egg Surface Membrane Complex......Page 303 2.3. Lectin Affinity Chromatography......Page 304 3.2. Solubilization of Sperm Receptor Complex......Page 305 4. Notes......Page 306 References......Page 308 1. Introduction......Page 310 2. Materials......Page 312 3.1. Operation of Freeze-Dryer......Page 313 3.2. Freezing......Page 316 3.3. Primary Drying......Page 318 3.5. Quality Indices......Page 319 4. Notes......Page 320 References......Page 321 1. Introduction......Page 324 2. Materials......Page 325 3.1. Prevention of Bacterial Contamination......Page 326 3.3. Use of Stabilizing Additives......Page 327 3.4. Low-Temperature Storage......Page 331 3.6. Stability Analysis and Accelerated Degradation Testing......Page 332 4. Notes......Page 334 References......Page 336 1. Introduction......Page 340 3.1. Gel Staining......Page 341 3.2. Electroelution......Page 342 3.3. Protein Precipitation......Page 343 References......Page 344 1. Introduction......Page 346 3.1. Electroblotting......Page 348 3.2. Protein Staining......Page 349 4. Notes......Page 350 References......Page 352 1. Introduction......Page 354 3.1. Sample Preparation......Page 356 3.3. Second Dimension: SDS-PAGE......Page 357 4. Notes......Page 359 References......Page 360 1. Introduction......Page 362 2. Materials......Page 364 3.2. Preparation of Acrylamide/Immobiline Partition Membranesat Desired pHs......Page 367 3.3. Loading Samples and Assembling μsol-IEF Chambers......Page 369 3.4. Electrophoretic Conditions......Page 370 4. Notes......Page 371 References......Page 374 38 Practical Column Chromatography......Page 378 39 Detection Methods......Page 390 40 Peptide Proteomics......Page 412 41 Multidimensional Liquid Chromatography of Proteins......Page 426 42 Mass Spectrometry......Page 448 43 Purification of Therapeutic Proteins......Page 456 44 Purification Process Scale-Up......Page 464

This second edition provides up-to-date protocols focusing on techniques for the specific enrichment of phosphopeptides and phosphoproteins. __Phosphoproteomics Methods and Protocols, Second Edition__ guides readers through different labeling strategies for quantitative phosphoproteomics; high-throughput mass spectrometry-based phosphoproteome analyses and phospho flow cytometry; and bioinformatics strategies for phosphoproteomics data analysis and integration. Additional protocols concentrate on the identification of kinase-substrate relationships by both high- and low-throughput approaches. Written in the highly successful __Methods in Molecular Biology__ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, __Phosphoproteomics Methods and Protocols, Second Edition__ aims to ensure successful results in the further study of this vital field.

Yeast Protocols contains many key techniques for studying the biology of yeasts at both the cellular and molecular levels. Working primarily from Saccharomyces cerevisiae, the expert contributors explain step-by-step how to successfully isolate, identify, and culture yeasts; the secrets of meiotic mapping; how to use PFGE in karyotyping and gene localization; the methods for purification and analysis of various cell components; and the construction and exploitation of genomic DNA clone banks. They also cover the latest methods for chromosome engineering, insertional mutagenesis by Ty elements, mRNA abundance and half-life measurements, the use of reporter gene systems, genotoxicity testing, and more. Yeast Protocols follows the widely applauded Humana Methods in Molecular Biology style: brief introductions putting the particular method in context, comprehensive lists of materials, cookbook style instructions, and troubleshooting notes to avoid common pitfalls and solve problems. The techniques can be used with confidence and success by both inexperienced newcomers and established researchers.

1 Cell Cycle Molecules and Mechanisms of the Budding and Fission Yeasts......Page 2 2 The Plant Cell Cycle......Page 31 3 The C. elegans Cell Cycle......Page 51 4 Developmental Control of Growth and Cell Cycle Progression in Drosophila......Page 69 5 The Xenopus Cell Cycle......Page 95 6 The Mammalian Cell Cycle......Page 113 7 Synchronization of Cell Populations in G1/S and G2/M Phases of the Cell Cycle......Page 157 8 Mapping Origins of DNA Replication in Eukaryotes......Page 167 9 In Situ Assay for Analyzing the Chromatin Binding of Proteins in Fission Yeast......Page 181 10 Application of Magnetic Beads to Purify Cells Transiently Transfected With Plasmids Encoding Short Hairpin RNAs......Page 189 11 The Cell Cycle and Virus Infection......Page 197 12 Methods for Preparation of Proteins and Protein Complexes That Regulate the Eukaryotic Cell Cycle for Structural Studies......Page 219 13 E2F Transcription Factors and pRb Pocket Proteins in Cell Cycle Progression......Page 239 14 Forkhead (FOX) Transcription Factors and the Cell Cycle......Page 247 15 Measurement of Geminin Activity in Xenopus Egg Extracts......Page 263 16 CDK-Activating Kinases......Page 279 17 Cyclins, Cyclin-Dependent Kinases, and Cyclin-Dependent Kinase Inhibitors......Page 291 18 Measurement of Wee Kinase Activity......Page 299 19 CDC25 Dual-Specificity Protein Phosphatases......Page 329 20 Assaying Cell Cycle Checkpoints......Page 345 21 Polo-Like Kinase-1......Page 355 22 The Ipl1/Aurora Kinase Family......Page 371 23 Purification of the Ndc80 Kinetochore Subcomplex From Xenopus Eggs......Page 383

This volume presents protocols on yeast cytokinesis that will help Molecular and Cellular Biology researchers in the use of these microorganisms to approach the study of general or specific key questions in cytokinesis. Written for the __Methods in Molecular Biology__ series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, __Yeast Cytokinesis: Methods and Protocols__ provides practical and step-by-step detailed protocols useful for a wide audience ranging from experienced researchers to beginners in the use of yeasts.

This detailed volume explores experimental strategies and protocols for the expression, assembly, characterization, and exploitation of transmembrane β-barrel proteins. Beginning with methodologies to study their assembly, the book continues with protocols for characterizing the landscape of transmembrane β-barrel protein interactions with other cellular factors, dissecting processes of protein transport in bacteria and mitochondria, examining structural characterization, determination, and prediction, and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Transmembrane β-Barrel Proteins: Methods and Protocols serves as an ideal guide for researchers seeking to expand our knowledge of these vital membrane-spanning proteins.

This volume will serve as a guide for students in the field of neurobiology, and be a bridge between basic science researchers, doctors, and surgeons in clinical practice. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Neurobiology: Methods and Protocols aims to ensure successful results in the further study of this vital field.

1......Page 2 2......Page 14 3......Page 26 4......Page 36 5......Page 52 6......Page 72 7......Page 92 8......Page 106 9......Page 118 10......Page 136 11......Page 150 12......Page 164 13......Page 174 14......Page 188 15......Page 208 16......Page 232 17......Page 250 18......Page 266 19......Page 282 20......Page 306 21......Page 316 22......Page 332 23......Page 344 24......Page 354

This volume provides readers with detailed protocols covering the main cancer cytogenetics techniques needed for clinical utilization and research purposes. The chapters in this book cover topics such as chromosome preparation for myeloid malignancies; chromosome bandings; fluorescence in situ hybridization probe preparation; array-based comparative genomic hybridization; and cytogenetic nomenclature and reporting. The updated reviews on chromosomal abnormalities in hematological malignancies are excellent guides for cytogenetics data interpretations and specific malignant diseases correlation. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

This second edition provides new and updated techniques and applications associated with synthetic biology. Chapters guide readers through the creation and regulation of gene circuits, manipulation of biochemical pathways, genome editing and modification, creating genome language and computing, as well as molecular assembly.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Synthetic Biology: Methods and Protocols, Second Edition aims to ensure successful results in the further study of this vital field.

Plant Virology Protocols offers for the first time a comprehensive collection of state-of-the-art techniques for generating transgenic plants that are resistant to plant viruses via the cloning and expression of the coat protein gene. Its unfailingly reproducible methods, perfected by hands-on masters, cover the entire process from virus isolation, RNA extraction, and cloning coat protein genes, to the introduction of the coat protein gene into the plant genome and the testing of transgenic plants for resistance. Methods for testing for transformation by PCR and Southern blotting, the detection of RNA transcripts by Northern blotting, and the production of protein by Western analysis are provided, as are methods for challenging the transgenic plants produced and for detecting and measuring the levels of virus. The authoritative contributors also discuss the history and mechanisms of coat protein-mediated protection and the key ethical issues surrounding transgenic technology. Unprecedented in its comprehensiveness and its many time-proven methods, Plant Virology Protocols is certain to become the authoritative standard reference for plant scientists who want to tap the enormous benefits this technology has to offer.

This volume collects a series of protocols describing the kinds of infrastructures, training, and standard operating procedures currently available to actualize the potential of stem cells for regenerative therapies. __Stem Cells and Good Manufacturing Practices: Methods, Protocols, and Regulations__ pulls together key GMP techniques from laboratories around the world. Written in the highly successful __Methods in Molecular Biology__ series format, chapters include introductions to their respective topics, lists of the necessary materials, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Inclusive and authoritative, __Stem Cells and Good Manufacturing Practices: Methods, Protocols, and Regulations__ will be an invaluable resource to both basic and clinical practitioners in stem cell biology.