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Current Protocols in Cell Biology 2010年完整版 5483页* X; _7 G% ^$ z( _2 a+ p
! m' v7 A& Q" ]1 p! n: n1 m/ u3 D
Online ISBN: 97804711430316 [6 V& w0 G5 C
DOI: 10.1002/0471143030
0 Z. m4 K4 y: Y, U, x/ p6 i/ W
Table of Contents
1. Preface5 ~! ]; ?- d2 y
2. Foreword
3. Chapter 1 Cell Culture
1. Introduction+ {! H+ l  A- s) W1 K+ Y
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture4 M1 [$ D4 D5 N! n1 k- b
3. Unit 1.2 Media for Culture of Mammalian Cells
4. Unit 1.3 Aseptic Technique for Cell Culture% s) C3 W' h, B- q- X1 B3 z8 H
5. Unit 1.4 Sterilization and Filtration
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
7. Unit 1.6 Media and Culture of Yeast9 S7 v- B0 Z( A6 j
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
4. Chapter 2 Preparation and Isolation of Cells
1. Introduction
2. Unit 2.1 Establishment of Fibroblast Cultures
3. Unit 2.2 Preparation and Culture of Human Lymphocytes
4. Unit 2.3 Preparation of Endothelial Cells+ v' H8 E: B' E' S6 d
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
6. Unit 2.5 Laser Capture Microdissection
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells9 N/ n/ r2 C3 p+ H" `+ P
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
1. Introduction
2. Introduction
3. Unit 3.1 Overview of Cell Fractionation
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains( _$ M# F- _0 x8 O2 q4 m
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient  t, W; x8 b# S6 ]
Centrifugation
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient0 i* c, U# ~& d
Centrifugation  R$ ]' V6 m# g6 r2 m0 ~5 W
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
Centrifugation+ J" p, ~2 F: d& M* Y
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
14. Unit 3.12 Isolation of Synaptic Vesicles1 J# `$ ^& R* y8 h, Z! {1 q
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation7 r' N( x$ \1 g& |" I6 M. i4 h
16. Unit 3.14 Isolation of Melanosomes
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
18. Unit 3.16 Isolation of Mast Cell Granules4 u7 E0 j% u. V! o; `( z  e- N, x
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster' i0 ^2 z5 @0 o, F3 z) \# _$ S. Y+ ]
20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas* Q( D. F  L9 N  ~8 z; N$ c
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons8 H+ J& $ E& i
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes0 k2 ^4 H8 M2 l
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
Fluids
25. Unit 3.23 Isolation of Intermediate Filaments
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle# T7 }1 b) [) G6 N+ S, r
27. Unit 3.25 Isolation of Myelin
28. Unit 3.26 Isolation of Renal Brush Borders' h( a4 U5 o( y& Z
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts0 i6 \+ H! H" ~
30. Unit 3.28 Isolation of Amyloplasts. j; R  t' q& r1 $ v
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
Centrifugation
33. Unit 3.31 Isolation of Neuromelanin Granules
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and  R8 l8 U1 |: n, E8 ]! _  H; ^1 f9 n
Density Gradient Centrifugation
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
37. Unit 3.35 Isolation of Platelet Granules- b% @1 D: a" W. @/ m5 ^
38. Unit 3.36 Isolation of Nucleoli* f/ K$ g) V6 S, l1 P8 E
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
41. Unit 3.39 Isolation of Chromaffin Granules
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
6. Chapter 4 Microscopy
1. Introduction' T! @( m' G$ e5 J+ J
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope( M. U# e! z% D8 ^! C& z% M
3. Unit 4.2 Fluorescence Microscopy
4. Unit 4.3 Immunofluorescence Staining
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
6. Unit 4.5 Basic Confocal Microscopy* B6 u' E* i2 _
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
8. Unit 4.7 Cryo-Immunogold Electron Microscopy
9. Unit 4.8 Correlative Video Light/Electron Microscopy' {7 i* N, _) p. u
10. Unit 4.9 Polarization Microscopy7 K. v, K; ^% h
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues+ `4 p" |5 X0 ^5 l
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
Events1 \  G; q9 P4 ~0 y; O+ Q5 f& r. t
14. Unit 4.13 Fluorescent Labeling of Yeast
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules  ^9 X  `; D1 Y& c
in Endocytosis: b! d! {( b& @$ E6 g
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images8 c4 q+ U: W9 H- G) B
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions+ I% S! s) P& d. E' `" T
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes$ x5 i$ o( @% k. p* Q1 w% W
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics3 }+ t7 z. r1 b) Z9 {1 T
24. Unit 4.23 Interference Reflection Microscopy' J1 R! \" D( M5 m1 W! j! i0 L
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
27. Unit 4A Organelle Atlas: Appendix to Chapter 4# q; J! V/ Y. C
7. Chapter 5 Characterization of Cellular Proteins
1. Introduction
2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein  N' V% n5 u; N1 }7 ]1 ]0 v# M
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients
5. Unit 5.4 Analysis of the Association of Proteins with Membranes( S" A" Z$ }* c$ O  ^
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
Spectrometry+ l. M& g( E4 W' Z$ z2 t
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening+ x6 {- q2 ?# U: Q( j$ }9 c+ z
Applications9 l; X; ^) f" u7 F% M
8. Chapter 6 Electrophoresis and Immunoblotting6 ?# J7 g9 H, S% w% a5 y( ~
1. Introduction
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins7 U6 |3 o; c3 A0 ?- ^9 [  S
3. Unit 6.2 Immunoblotting and Immunodetection
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
5. Unit 6.4 Two-Dimensional Gel Electrophoresis5 x' C. W- {. }, H
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
7. Unit 6.6 Staining Proteins in Gels
8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots& E) Q5 Z4 U; o! b7 M
10. Unit 6.9 Digital Electrophoresis Analysis
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis8 a9 W/ F5 o- @; S7 U( v$ g6 t- M
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of8 N$ q! n" j" c% {
Single Cell Gel Electrophoresis (Comet Assay)0 t+ D/ K) `4 a7 B. I# c
9. Chapter 7 Protein Labeling and Immunoprecipitation3 d% S( h, K$ K
1. Introduction4 Q2 B: L" ^) U3 S  Q% j
2. Unit 7.1 Metabolic Labeling with Amino Acids3 L% @' V* e6 ?0 P# A* T
3. Unit 7.2 Immunoprecipitation3 k( ?- ~: [, G  q, n+ Q
4. Unit 7.3 Metabolic Labeling with Sulfate
5. Unit 7.4 Metabolic Labeling with Fatty Acids% H1 c( Z! s/ F4 J# D' r. B' b
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins( k  ^. u: @+ U8 \
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars+ I9 a! N/ d6 `0 r4 K
10. Unit 7.9 Analysis of Oxidative Modification of Proteins' @- C/ P" s$ ?
11. Unit 7.10 Radioiodination of Cellular Proteins5 Y, F) {3 F) I0 j. I- j
10. Chapter 8 Cell Cycle Analysis
1. Introduction
2. Unit 8.1 Overview of the Cell Cycle* o% Z$ {, V$ `6 s% M
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells3 y* ?' Z: Y4 Q, b/ Z
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
11. Chapter 9 Cell Adhesion8 a6 A* N- r* \5 M9 L- B5 R/ P
1. Introduction% R7 |* y% D  |$ n9 [! z0 Z
2. Unit 9.1 Cell-Substrate Adhesion Assays
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force) T+ F- c7 N- J+ B8 R$ V( a$ u
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion( W" p4 I7 T9 s6 h6 A+ G" V+ @
5. Unit 9.4 Analyzing Integrin-Dependent Adhesion5 J% b, b8 R) d3 w
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules; ?4 {2 Y0 I* _, m: _
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions7 Z6 I8 m0 C. S9 q* l
12. Chapter 10 Extracellular Matrix' x# s5 u' m9 Q
1. Introduction* _& M6 ~  n: z& V: M: ]( l+ Y
2. Unit 10.1 Overview of Extracellular Matrix! H# h5 l& M9 i" a
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
4. Unit 10.3 Preparation of Gelled Substrates1 R1 u% y4 u7 C+ L3 k
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9$ C2 y4 \! q6 t- F  r7 ?% K4 h
Endodermal Cells
6. Unit 10.5 Purification of Fibronectin
7. Unit 10.6 Purification of Vitronectin
8. Unit 10.7 Proteoglycan Isolation and Analysis- E( X9 T1 P2 @# D, z. D
9. Unit 10.8 Matrix Metalloproteinases
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts6 Q& e# e& `" \- N6 t
11. Unit 10.10 Purification and Analysis of Thrombospondin-1+ r# s$ [& I# t) ^
12. Unit 10.11 Purification of SPARC/Osteonectin* G( J- a7 g1 J/ ^' n+ u! {
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly# e0 Z% q  T3 W1 T
14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor8 o, A6 C, m( Z6 U* O5 b
Xenografts
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning& g  t, e4 n, E: V" q9 W4 @
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D* @% J2 X) ~0 f1 {0 V$ V# \0 E
Cell Migration; R& ^: w$ \/ D) y, @3 r
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
13. Chapter 11 In Vitro Reconstitution5 t( d0 I/ l' e3 `3 U$ W
1. Introduction! D0 v4 m0 B& k, a& A1 Q. p+ J
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems# u- e4 \7 q" ^0 P* z3 h) G
3. Unit 11.2 In Vitro Translation5 {- V/ d. F0 `) D- @* W
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication* D& U" s0 U$ z/ V
7. Unit 11.6 In Vitro Transcription0 v' Z! T8 j+ c( c
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
9. Unit 11.8 In Vitro Translation Using HeLa Extract, O; g5 C- M; i4 ]; Z  W5 v
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts6 ?' C; d9 l4 X8 v: b
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts  k2 N6 @! i" L2 F
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import( M& H; F2 p9 Q8 j$ `
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts1 Q; g, B/ r, n1 n& D8 ]
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
14. Chapter 12 Cell Motility
1. Introduction& b9 P; f& F* t% S% E, m2 c; l1 J
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
3. Unit 12.2 Invasion Assays
4. Unit 12.3 Cell Traction6 \8 }! _2 e( m+ o, z
5. Unit 12.4 Cell Wound Assays+ N: ]9 K3 m) R' H  i
6. Unit 12.5 Dictyostelium Cell Dynamics6 f' y* v3 k/ g& A( B# [
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils2 W, S# W9 S: Q  ?) i/ y3 C
8. Unit 12.7 Actin-Based Motility Assay
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP/ d8 b- y" c; S7 r
15. Chapter 13 Organelle Motility* ^  m3 K6 Q5 v7 K: E0 T1 j
1. Introduction) k7 G5 P- u( \( [
2. Unit 13.1 Microtubule/Organelle Motility Assays
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin# ^* n( j; n; J' [/ Y0 Y
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
5. Unit 13.4 Movement of Nuclei, ~9 M& y8 M% I
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching7 \1 C) D& G0 {9 w; d3 Q. e2 N
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly* I/ u( q- V. ~' `( L$ `
16. Chapter 14 Signal Transduction: Protein Phosphorylation
1. Introduction
2. Unit 14.1 Overview of Protein Phosphorylation8 s( w7 x9 L; E7 g) ]3 X9 z0 c! E
3. Unit 14.2 Immunological Detection of Phosphorylation- I& c+ H  B6 ~$ t
4. Unit 14.3 The Detection of MAPK Signaling
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation8 w& j: M. x8 q( ^/ n3 H
6. Unit 14.5 Phosphoamino Acid Analysis
7. Unit 14.6 Determination of Akt/PKB Signaling$ Y' h) |# G6 @1 t- a
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events
9. Unit 14.8 Rho GTPase Activation Assays
10. Unit 14.9 In Vitro GEF and GAP Assays, y$ V9 S6 f3 D0 t3 l, |3 z
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
Energy Transfer (FRET)5 N- O1 C% m* t) t1 F6 W
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells8 P: k  u, y1 n- T" w
17. Chapter 15 Protein Trafficking
1. Introduction/ E. e; A' J" ^* s/ y
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways7 k1 A) r( n( X- T3 C& S5 M
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
4. Unit 15.3 Endocytosis: Biochemical Analyses8 G) i% k8 R+ B4 l- ?2 q/ {- ]
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane7 p5 v7 r  ]- V+ z
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells# n4 R! {7 @2 G0 U
7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
9. Unit 15.8 Analysis of Protein Transport to Lysosomes
10. Unit 15.9 Studies of the Ubiquitin Proteasome System
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network; Q7 Y; m$ i7 n2 V6 Q, K; Q1 `$ k
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells- ]) y( L# Y) I( N: r2 Y/ W
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
EGF Receptor
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
18. Chapter 16 Antibodies as Cell Biological Tools* X1 p$ s3 Q$ p; G0 E. f7 o
1. Introduction
2. Unit 16.1 Production of Monoclonal Antibodies% f1 ~8 H: |0 A. X
3. Unit 16.2 Production of Polyclonal Antisera
4. Unit 16.3 Purification of Immunoglobulin G% g5 e+ k- Z" u
5. Unit 16.4 Fragmentation of Immunoglobulin G
6. Unit 16.5 Antibody Conjugates for Cell Biology; [0 Z! _8 ?  I
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
19. Chapter 17 Macromolecular Interactions in Cells+ n# i0 r1 c, v* t
1. Introduction% `! }+ I- b3 i- A- X# A. r4 L1 K
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)3 n  N( f- }5 b& n" [
Microscopy
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis7 w, m5 [" Q: R! _7 s* b
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins7 x# U$ q" c1 V$ m1 y( C
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries& y$ J3 D9 g1 r" b- U9 i, C, z4 j# ^
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific! U4 o; F+ C  f  C/ d* P
Genomic Sequences In Vivo2 i  g, O2 F! M/ l
9. Unit 17.8 Isothermal Titration Calorimetry
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking8 ^* }% V* e  n. b+ r  G8 s
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
Interactions (RFAP) in Live Bacterial Cells
20. Chapter 18 Cellular Aging and Death
1. Introduction
2. Unit 18.1 Current Concepts in Cell Death. o2 d7 p$ j+ ^+ G; A5 @2 g
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis+ c" z' B. R# W3 X2 d
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria4 S8 j2 J& U5 A" A! K  ]
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
7. Unit 18.6 Analysis of Telomeres and Telomerase
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of0 l& |, [2 r2 p/ P' n5 ?1 D: i
Poly(ADP-Ribose) Polymerase) q; t3 j, i" s5 v# i9 @2 D3 w
9. Unit 18.8 Flow Cytometry of Apoptosis
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase
Assay% I1 d' c9 d" Y/ P3 ~/ q
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis. Q. t, \" V& f: o
21. Chapter 19 Whole Organism and Tissue Analysis
1. Introduction1 x$ Z9 b3 x/ s- d* Y
2. Unit 19.1 Overview of Metastasis Assays
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene! p: K4 C. F7 ^1 W' n( \5 m+ h
Expression After High-Fidelity T7-Based RNA Amplification
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA  I% V" I; E0 a. w( Y+ ~% y$ B
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
9. Unit 19.8 Embryonic Organ Culture
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin2 Y1 K: I3 S" ~: ~
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
12. Unit 19.11 Generation of Transgenic Mice( V1 w4 Y: r" G2 U
13. Unit 19.12 Overview: Generation of Gene Knockout Mice
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
22. Chapter 20 Expression and Introduction of Macromolecules into Cells
1. Introduction1 C4 K+ A6 V. e. y9 _7 ]! E2 I
2. Unit 20.1 Direct Introduction of Molecules into Cells
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
4. Unit 20.3 Calcium Phosphate Transfection
5. Unit 20.4 Transfection Using DEAE-Dextran
6. Unit 20.5 Transfection by Electroporation9 e' V6 B6 S1 ]- F3 J6 @" D
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents  h. ^1 z- s1 b# G
8. Unit 20.7 Optimization of Transfection
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
23. Chapter 21 Fluorescent Protein Technology
1. Introduction3 u. s* G4 V1 a* D& E0 F
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence) ~. C+ Y1 M5 h! R
Complementation (BiFC) Analysis: L) p1 z, z- u( {: B
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
6. Unit 21.5 The Fluorescent Protein Color Palette% i6 m- o" t4 y  q1 @4 r# s/ U; `
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins" n9 s, |' q: h6 ?) j/ B0 T/ e% W
24. Chapter 22 Cell Biology of Chromosomes and Nuclei& `1 [% C9 i, q, ^
1. Introduction6 d$ |% s( X, Y& R+ u
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples$ w1 z# S; D/ v7 x3 B7 [# v3 k& U
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis5 P* P" \- n6 A0 ]/ S
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)) z& x) x2 n) W5 N7 v2 ?7 p( ?
6. Unit 22.5 Multi-Color FISH Techniques0 I& }2 e3 j8 |, f. Z
7. Unit 22.6 Comparative Genomic Hybridization
8. Unit 22.7 Sister Chromatid Exchange& ?  A; h7 O0 r+ P
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes* w% |, M( _4 U: p- R* _7 X/ G
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
14. Unit 22.13 Monitoring mRNA Export
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-
Field Gel Electrophoresis% N1 U3 U, {* R: M( C& f' `$ b1 v5 V
25. Chapter 23 Stem Cells5 @! t$ x5 A( d& b/ U" s  C
1. Introduction
2. Unit 23.1 Stem Cells: An Overview
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture7 C; {/ S$ R# P8 q' W. X+ Z! ^
and Differentiation as Embryoid Bodies
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
Vessels) b- W+ [) {( }/ B% g$ O
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into/ y, G& S% Q6 N# c9 x, e
Adipocytes
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation& ?( D+ w, ?0 ^/ g
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells* w9 z/ c$ W* W  x  L! F8 W
8. Unit 23.7 Neural Differentiation of Human ES Cells
26. Chapter 24 Lipids4 v: x# E, _2 ]; w
1. Introduction
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
27. Chapter 25 Nanotechnology$ T. K2 L1 o9 a% A' O
1. Introduction
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes. ~+ n- q% U! y, |
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering2 M/ Y; b8 A( q) L* \
28. Chapter 26 Viruses: S0 o3 ~% p% N6 \2 j8 d
1. Introduction& Y& R3 y' ^" G
2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors. M- O. u1 [& ~& g% H
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of2 a0 ~: E1 S( q  G7 v4 v
Cell Entry
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses; P1 o+ ?2 b7 N: h2 Q% m! k/ I
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission* T' M) ~4 H+ e$ n% w! [
29. Chapter 26 Lipids0 K3 Z, @4 K0 Z
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by- |# F2 ~$ [, U
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
30. Chapter 27 RNA-Based Methods in Cell Biology
1. Introduction% ^" H( Y+ U5 v3 V
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible1 B8 |& D. E+ r$ c7 }
Rescue in Mammalian Cells9 _1 i! y4 P7 L, F) c- R
31. Appendix 1 Useful Information and Data
1. 1A Useful Measurements and Data
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research6 `8 _) S- p) p* W# q+ c8 F
3. 1C Identification of Motifs in Protein Sequences- j1 C4 q( X7 E/ E2 d5 K
4. 1D Safe Use of Radioisotopes! F2 u& Z3 S8 n
5. 1E Absorption and Emission Maxima for Common Fluorophores- [+ c9 R( {( M* T
6. 1F Importing Biological Materials; s6 L" {7 y7 q+ V
7. 1G Centrifuges and Rotors
8. 1H Internet Basics for Biologists
32. Appendix 2 Laboratory Stock Solutions and Equipment1 x0 ~  r* @0 V0 _+ r; [  @
1. 2A Common Stock Solutions, Buffers, and Media
2. 2B Medium Formulations
3. 2C Standard Laboratory Equipment7 W+ r; X% G2 m, x( ?
33. Appendix 3 Commonly Used Techniques
1. 3A Molecular Biology Techniques
2. 3B Spectrophotometric Determination of Protein Concentration" k$ n5 e2 z5 B* h" u
3. 3C Dialysis and Concentration of Protein Solutions( }1 B  `' L% W
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy$ A: h, \3 C4 T/ h; L
5. 3E Silanizing Glassware0 K' Z. x, x0 {6 ?& z4 J
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
7. 3G Micro RT-PCR5 d5 D, s& j% N
8. 3H The Colorimetric Detection and Quantitation of Total Protein
34. Appendix Suppliers" x' W0 k0 i0 z: y5 ?) B+ O: C
1. Selected Suppliers of Reagents and Equipment
' \0 D! C; F  D/ J3 @

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