标题: PDF电子书：Current Protocols in Cell Biology 2010版 [打印本页] 作者: cz200203 时间: 2011-3-8 15:53 标题: PDF电子书：Current Protocols in Cell Biology 2010版
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Current Protocols in Cell Biology 2010年完整版 5483页 ; d6 |% O( [1 {0 ] e9 m0 D M6 m1 R9 H% i# d: e2 h# J1 P; }
Online ISBN: 9780471143031 1 y5 h) R& @8 N/ }, Q% L5 lDOI: 10.1002/0471143030 / D* u' {" L2 A5 U i( _9 g' G1 R& FTable of Contents 7 X! |; J- `# U$ ]1. Preface 2 D5 r4 Q; P- ^# w- h4 b% f2. Foreword3 C, m; v3 t$ q$ ]- V% c
3. Chapter 1 Cell Culture + M! e4 r0 W- R" ~1. Introduction- m& Z; S+ F x* a6 w3 r& m
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture 4 @! d0 O0 [9 @% b+ F9 u- \. l3. Unit 1.2 Media for Culture of Mammalian Cells & E* ^! e/ E- X4. Unit 1.3 Aseptic Technique for Cell Culture- |' {( x- G$ K# E9 `, I
5. Unit 1.4 Sterilization and Filtration 3 x) x r9 M9 \1 u# l6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures# l# ^8 w7 F9 |$ x5 e1 r- y
7. Unit 1.6 Media and Culture of Yeast 5 J# f7 ^. ]+ t- b+ b8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging , m+ f8 E3 Y. i h2 B$ c4. Chapter 2 Preparation and Isolation of Cells9 j' S, x5 }/ T# I* y8 k2 i
1. Introduction* z3 H/ E( b4 p
2. Unit 2.1 Establishment of Fibroblast Cultures0 X2 Y& X0 }9 F% W) Q' f
3. Unit 2.2 Preparation and Culture of Human Lymphocytes0 |( Y( K3 ], d% e( s" p1 J7 H* H
4. Unit 2.3 Preparation of Endothelial Cells* k6 G7 D6 X' e2 m* f0 q& b: z
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation 5 _* }( t5 C- g6. Unit 2.5 Laser Capture Microdissection/ K! J+ w6 p. X/ I. l3 w$ q' K, r
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures * V" l) h+ }# q" v+ c) V/ g8 e* i3 k8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells1 @9 d- r K2 F8 L5 X1 F
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles& |) W9 I2 {4 C2 [
1. Introduction 7 E7 {& Q7 F8 X# e0 Z; @2. Introduction! X% Y: f+ S; w" s6 V8 F
3. Unit 3.1 Overview of Cell Fractionation8 q6 V0 I+ U8 U4 L. b7 _" r
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains: o5 @- L2 b$ ~& k
5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation0 L$ @' R. k4 j7 O: l; f& x
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation + _. C% \ J0 G' o$ ?' V5 x/ r7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient $ Q4 {! U8 o$ l$ t# |1 FCentrifugation: i- L0 a2 B8 g; e# R6 y z
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient A) h o' y4 R8 t' x
Centrifugation) g+ g o- ~5 Q+ ^
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae % n% a* t$ U' y; _. D+ m1 P# v10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae8 Q9 l9 x2 t) }2 e
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient1 i# W0 K! b. e) a4 e: o
Centrifugation 2 u& T+ j& I v/ N/ l12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues & V% X# P2 u5 I2 U8 G13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes $ h) y% v( p6 W" `8 i: b7 _ E C" j14. Unit 3.12 Isolation of Synaptic Vesicles 2 m2 S4 Z7 F1 a; A15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation! x+ y% @9 y# x6 q- Z, Y
16. Unit 3.14 Isolation of Melanosomes ! F6 {; p% O. M17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation 3 ?3 V9 x H/ G3 e# a18. Unit 3.16 Isolation of Mast Cell Granules& l6 S7 ~1 F Q. S0 w
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster , s# k; N1 ]8 l( c20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas 3 P" i. i, ~* O21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons , {( F; x9 e, v6 Q22. Unit 3.20 Isolation of GLUT4 Storage Vesicles6 Q$ B8 o" b: s0 l* Z. J
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes 7 M. Z$ N; G) k6 C5 v+ i6 n% {! T24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological ! Q% \, M3 D* I0 u+ A! l Q0 |: e1 T( ^Fluids % V: \8 J+ a# [7 B3 l0 Q: L) @25. Unit 3.23 Isolation of Intermediate Filaments- U7 N9 U9 K9 Y5 T
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle! q: e7 Y: G8 l% d3 U7 R8 y3 C1 x) V
27. Unit 3.25 Isolation of Myelin 4 w1 M& _" m; M! g" C" \) F/ S) W28. Unit 3.26 Isolation of Renal Brush Borders / J+ A' {, M" I0 @29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane4 u* W! ]4 X, B- [1 Y
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts " j% {/ P) b5 \9 e0 r* h30. Unit 3.28 Isolation of Amyloplasts+ o9 w4 R/ n. W/ b" P: e; y* r& V
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins1 M- F8 u2 W& p$ z, O2 v& C* {
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic , s2 d2 B8 r1 a6 sCentrifugation1 R5 ~$ o- e3 Q* }2 }
33. Unit 3.31 Isolation of Neuromelanin Granules - Q1 B* C4 l) S0 s( j( X% X34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and 3 R+ T2 c7 C' bDensity Gradient Centrifugation( Z2 s/ c5 A5 w1 D. I
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments 0 z/ }# e( x. h0 d% }) y/ E/ W36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation + x+ U$ p, ^+ R5 A37. Unit 3.35 Isolation of Platelet Granules7 z5 ~, f. F) n5 s4 r( i7 {* {
38. Unit 3.36 Isolation of Nucleoli6 H& M1 V4 p9 e8 Z" a
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins$ Z) B( q. Y7 |; n
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates % V h0 L. ]5 D5 y9 E41. Unit 3.39 Isolation of Chromaffin Granules5 B+ K) f% A8 K: W& K5 V# V
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines ; U6 a3 V! h. T1 d8 W( W7 ^- N5 k# Z6. Chapter 4 Microscopy - j* z8 |2 w* q- Y$ R) |. Q1. Introduction. Z/ T' u' a' p' g
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope) G$ E3 h% |, ^' O* j; K- }
3. Unit 4.2 Fluorescence Microscopy 7 F9 i- w+ T2 A: x7 N+ S% L! ~4. Unit 4.3 Immunofluorescence Staining6 w1 R. L9 y( v+ d
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria; ]. D0 w4 j$ h1 A
6. Unit 4.5 Basic Confocal Microscopy - Y! P/ m( H$ p' l) U7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues 7 Q' c* D! d) u5 j% K' |* L. Y8. Unit 4.7 Cryo-Immunogold Electron Microscopy# X! e# Y3 D' D
9. Unit 4.8 Correlative Video Light/Electron Microscopy 2 C# M8 H6 k! |9 F# @, a10. Unit 4.9 Polarization Microscopy! C+ l) S8 @) a* M) L
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells b" P9 V0 V8 a/ \/ A) L b; v# ~! Z
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues , d$ ^+ Q& t0 X t% ^. [13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface 9 F2 f. }5 G; k* E% t" gEvents ! l+ T7 _5 R1 O; V14. Unit 4.13 Fluorescent Labeling of Yeast( ]5 d3 Z# L( Q% f
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy 3 M% t0 K* F$ B" d16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy 9 l% ~+ t3 c! @, `17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules2 V1 f, F, L5 C5 Y7 f q% Q: I
in Endocytosis6 ^! f, o- g: L; u* `! B
18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology ' E4 f- E) R# v: {( { q19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development Z: a9 O; B3 r" K: E; V t% ~
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images @- t+ Q( B" z6 D! N
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions; G0 t$ r( q+ I& W% P% M9 J
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes( l+ D/ u9 g: h8 ]! y/ i2 J- f5 e
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics/ @5 r+ r' u- m; ]) v8 T
24. Unit 4.23 Interference Reflection Microscopy9 y+ F+ ~& c5 U" n$ ~& c
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach * ^. k2 ]) F+ E; @% U6 n) y' m( h" X* o26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches $ Y& P8 Z/ d3 n3 z* s27. Unit 4A Organelle Atlas: Appendix to Chapter 4$ m+ h* X0 g' a! f0 }
7. Chapter 5 Characterization of Cellular Proteins ) [' H) \# X8 w, _1. Introduction 4 H1 c+ f6 i5 y2. Unit 5.1 Overview of the Physical State of Proteins Within Cells: F9 X2 U! q+ T! O% S# I _
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein4 p0 P9 n" q4 C9 u% k6 \8 u
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients : E5 b4 S- [ }& E+ j5. Unit 5.4 Analysis of the Association of Proteins with Membranes4 l* w& m/ j+ c9 m9 f, w5 R
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)% D3 i" q. V* B- D8 ]3 H; f
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass M! Z) o6 R6 R4 {, g" X P% C
Spectrometry2 j% ^6 c+ |" c/ N. m
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening1 g% Q8 W s; L" I
Applications4 A& M! C. X$ C, z
8. Chapter 6 Electrophoresis and Immunoblotting3 a* ~9 {9 J/ u
1. Introduction ) i6 w" ]4 Q. Y- M; E1 v2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins 0 ^+ W0 j3 y4 T# [3. Unit 6.2 Immunoblotting and Immunodetection 4 }: |* `2 b8 Q" O- A4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots - J# \7 V) e4 w: Q5. Unit 6.4 Two-Dimensional Gel Electrophoresis. i. ]6 d2 j7 c. r# i* X6 g+ {
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions " P9 Z A7 O1 F( H0 G7. Unit 6.6 Staining Proteins in Gels L( x* c I% q4 H# ~8. Unit 6.7 Agarose Gel Electrophoresis of Proteins3 M3 X) O$ w' h7 X5 w
9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots, y' w) j: p7 B2 t |# L; o3 |
10. Unit 6.9 Digital Electrophoresis Analysis- z4 K, j& Y+ B9 U
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis & V. T. A$ {0 W; ]% Q4 G12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of6 c7 }- I# ]! U$ T* {
Single Cell Gel Electrophoresis (Comet Assay) 9 Y6 B S. j6 y- G4 h/ e9. Chapter 7 Protein Labeling and Immunoprecipitation 0 p" X5 Z8 O+ ] N* D1. Introduction/ ~; H. A, b: _" W2 ^9 e! K/ m
2. Unit 7.1 Metabolic Labeling with Amino Acids0 X# B5 j4 t- o4 z
3. Unit 7.2 Immunoprecipitation ; z$ r. R4 ^6 A1 G4. Unit 7.3 Metabolic Labeling with Sulfate& f4 O" z9 x. I& Q' t* u
5. Unit 7.4 Metabolic Labeling with Fatty Acids # }! { q$ n$ R! p9 Z1 m9 C6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups8 X" R" W. ?+ Z* e/ d' N6 W" C1 `
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins 5 N3 A/ B; n) u! Q* v8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins & e' s! s# P: L) K9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars3 }; G3 y/ H& z5 z2 f! d
10. Unit 7.9 Analysis of Oxidative Modification of Proteins1 g3 K& W- W* G5 }/ m
11. Unit 7.10 Radioiodination of Cellular Proteins 5 P+ `# i& j- {, {+ c3 M& Y10. Chapter 8 Cell Cycle Analysis , ^5 g; m0 g6 p8 w6 d2 t1. Introduction5 w4 S; ` ~3 E% n
2. Unit 8.1 Overview of the Cell Cycle& k4 K j& k2 {. @6 o w
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle, @+ `6 @; |+ M- A, X
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle ' t. y; E, P( C6 ?- c8 `" Z9 Q4 Z5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry0 z) A" ?0 V" `' C' f
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells/ W/ _, Y7 o+ j' H
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry # @* |5 A# g8 p4 {$ ^# P11. Chapter 9 Cell Adhesion3 t7 T2 b8 d4 j4 r# C2 T9 Z
1. Introduction p- ~+ P+ h& ^# `3 G
2. Unit 9.1 Cell-Substrate Adhesion Assays% M3 t v% B3 K) k% Q
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force6 W8 u( x- |* n* N$ z
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion ' o$ ^+ c" a0 V5 \, [5. Unit 9.4 Analyzing Integrin-Dependent Adhesion : _7 v2 U! S ?# |" Y. M6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules' H: N% e9 X! s1 E+ f7 C
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions ! a" {! J4 e2 f% ?% |% U12. Chapter 10 Extracellular Matrix: U0 @6 L, d! k/ N9 I
1. Introduction4 r9 F. u- h- ^3 E8 U8 K
2. Unit 10.1 Overview of Extracellular Matrix ! ~0 a) c( y7 `! e q( B3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors & z& W/ F1 U# L% D4. Unit 10.3 Preparation of Gelled Substrates& }! y4 V6 {. _8 \0 w
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9 2 o( Z2 y/ J8 W) g2 ]6 ]% O9 F+ U [Endodermal Cells/ N8 s. T {; z8 i* D! o" i
6. Unit 10.5 Purification of Fibronectin * S0 Y _: ^; \5 f" V7. Unit 10.6 Purification of Vitronectin. l% S: d) j8 Y- c, F
8. Unit 10.7 Proteoglycan Isolation and Analysis 4 B- m0 [% K; N6 q3 o: e9. Unit 10.8 Matrix Metalloproteinases $ C7 N6 a2 a2 r' d7 ~10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts # e# c$ Q8 x- C' j3 \3 d11. Unit 10.10 Purification and Analysis of Thrombospondin-1 x2 f+ x# w6 Q; P; X/ v12. Unit 10.11 Purification of SPARC/Osteonectin) _; C3 O. T ^6 s) M2 R
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly . J P, O2 u2 A8 Y14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly8 _1 D* d. G0 k6 S( y6 q
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor9 z4 K R& ]( ], i1 a; g' T! X/ ~
Xenografts 7 t4 a' \" q+ u16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning7 G+ k8 f9 U; }0 ^4 u
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties! |/ O: v8 u; S* W8 O- N7 @
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D, [! S X3 {3 n1 h; J
Cell Migration+ [( r; j0 h& v6 f7 r+ n& p) M& H
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix. y* i$ a$ l, J, v' C
13. Chapter 11 In Vitro Reconstitution - I m. d. s- ~1 I! ?8 B1 ?1. Introduction " i& y A/ q: c! y0 E/ V. D8 _2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems2 J; g A6 P1 E a/ J) {- @3 v
3. Unit 11.2 In Vitro Translation1 Q2 m6 |& Q- [" P" t6 L
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells 5 Z2 L3 c) F8 y4 b5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes4 k+ J3 l: c+ D& I( W8 ~' k1 Q# z
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication: B+ S* { `+ e* ^3 K3 w
7. Unit 11.6 In Vitro Transcription / M! ~/ j/ b9 P( y+ X' e1 S: ?8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells% n/ C C! O2 J5 Q- \. u% l {3 U7 M
9. Unit 11.8 In Vitro Translation Using HeLa Extract9 M6 x5 F* {! b* E8 w8 c- P
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems + E- V/ k, [: Z' j8 B7 k11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts9 u4 ?. |# }0 g5 D
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts6 X" q" R' a( e+ z
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts- |$ ^; R" F/ t' p1 j2 B! `# k- n
14. Unit 11.13 Mitotic Spindle Assembly In Vitro% K/ r4 ^9 s5 {8 \
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes' }. ?5 A! G- ^2 K, e0 n( ]3 j
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import/ V, `9 q; N; E$ r8 y/ P
17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import: H! e' R2 m6 j9 k( M1 v! S* i
18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts ' H% S" ?2 o6 A6 C19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells& ^0 w) V9 F+ r5 L! j- I
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import7 T, x8 B6 w! F: U
14. Chapter 12 Cell Motility; H/ [$ R; Z+ H) d2 T _
1. Introduction" j" n( `* M# x" P$ K' L' b$ j
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells9 d# @7 }: a( W( H# o z
3. Unit 12.2 Invasion Assays ) B4 H! m* Y4 ^$ s) \4. Unit 12.3 Cell Traction " I! q4 u; b) h5. Unit 12.4 Cell Wound Assays# _3 f0 S; S6 d- c( x
6. Unit 12.5 Dictyostelium Cell Dynamics 2 T" O7 S* A' q: ~6 u& E7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils - D0 n9 P. T$ T6 f+ M4 Z: v8. Unit 12.7 Actin-Based Motility Assay ; K2 D( E: Q8 _$ j1 H9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP 0 u0 Q/ T* V% A* P, \15. Chapter 13 Organelle Motility, ] X2 y: Q7 }2 x6 \) C
1. Introduction - N, a0 Y: I# V' F: O3 |5 D2. Unit 13.1 Microtubule/Organelle Motility Assays " ]% C1 [3 W, {3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin* \. r u# k' o1 s- h4 ?) d8 s* V3 l
4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP ! e- \# u0 H$ q \/ U5. Unit 13.4 Movement of Nuclei) ~# @3 T' S6 L. ^/ `- T3 x$ z0 L+ Q
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching 5 e) Z# r' Z5 g. G1 t7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly0 |/ E! u! }5 }, P2 s
16. Chapter 14 Signal Transduction: Protein Phosphorylation 9 y! v( d1 O% Q+ i# t" g! P- N1. Introduction ! x3 c/ U( s% `6 H. ]2. Unit 14.1 Overview of Protein Phosphorylation 9 U3 t6 |' y) n. x3. Unit 14.2 Immunological Detection of Phosphorylation $ c8 z- I g0 b7 y" d5 [0 x4. Unit 14.3 The Detection of MAPK Signaling& h- ?. G: B! z+ I% V
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation . }* r% C7 S' i6 [6. Unit 14.5 Phosphoamino Acid Analysis! T# ?* i' \( Z0 s1 [% m/ O
7. Unit 14.6 Determination of Akt/PKB Signaling . T- T! \- j& t l* M4 v0 l8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events/ y& ^5 ], H/ H7 X9 E% `; o) ]
9. Unit 14.8 Rho GTPase Activation Assays3 {, U* v. r% n% x w3 Z, }
10. Unit 14.9 In Vitro GEF and GAP Assays 2 E3 I, J* Q) H9 f K6 _! F6 C11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance , d, J! Y8 P& kEnergy Transfer (FRET) 5 ~6 b3 `! n9 U& `0 v; }) d12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells C1 ^$ e5 t' O% n7 @3 K! T# z13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells % R3 G! G7 B# d17. Chapter 15 Protein Trafficking* B: {1 S- N9 {6 g6 Q; ^1 i5 r
1. Introduction , v6 p$ E) w5 v4 L2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways3 k; i1 j! C( c3 R
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking% r6 l% u, v% N4 |/ w
4. Unit 15.3 Endocytosis: Biochemical Analyses% C. z* c( W: A- m
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane 2 s+ |7 |( ~, ~1 { f! n: D! x# Q: b6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells / m0 y( z( K) W6 `. i6 g7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum 1 F/ ?1 z* c7 K0 V) S8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation; g( D! n! y, Z0 t C- g
9. Unit 15.8 Analysis of Protein Transport to Lysosomes: i2 ~0 E' H! {% m" s7 f
10. Unit 15.9 Studies of the Ubiquitin Proteasome System$ L# J; f6 p, d
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network 2 K# _: W6 X: X! t12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules * H( M' a' `& }13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells! i9 l# s- K4 w! O, w
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging9 R2 m }4 | i3 I1 j& u5 K
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and % G6 Z9 ^1 R2 b# cEGF Receptor" i5 s) G: v$ Z: l2 L* N& G
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers 3 [0 V+ n6 i! O* ]* u& l18. Chapter 16 Antibodies as Cell Biological Tools 6 w$ ?* N& p! ?' A3 A1. Introduction( Q" J- H* a3 e5 [
2. Unit 16.1 Production of Monoclonal Antibodies; v5 F# S$ A6 ]* N; `0 @- T8 V
3. Unit 16.2 Production of Polyclonal Antisera1 T3 W1 D2 q5 o$ ^
4. Unit 16.3 Purification of Immunoglobulin G2 b( T( o# J6 z* U) I R# k$ I
5. Unit 16.4 Fragmentation of Immunoglobulin G+ h; r3 H: u0 ]! `; j+ l8 p
6. Unit 16.5 Antibody Conjugates for Cell Biology ! M" P c$ J. z4 e5 L7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides , f2 f! U/ y! E% D0 y8 V19. Chapter 17 Macromolecular Interactions in Cells! _% q) v8 K& K1 o
1. Introduction% P: |4 y$ T+ h; m. J: J
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)5 `1 L V3 F# a" {" p
Microscopy , ]' m, p& o5 s8 u. E+ E3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis" e3 R- } Q& x8 E5 w( ]
4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins# ?% a1 E9 X. P! f" H
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries 5 I/ E3 ]# H& y2 R; S6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry 4 A# J: g. ? P% X8 G# `7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors. \$ u) a/ W6 K
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific ; x7 N! S. J$ s5 A6 |: s2 z. NGenomic Sequences In Vivo% R1 M$ ?% d: N4 e) O+ |6 Z6 V
9. Unit 17.8 Isothermal Titration Calorimetry ; x e7 X# C7 X, ^9 X10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells 3 [9 |& g# I7 n3 k; Z( |- a11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking9 l& W; u& |$ I8 e. s E+ D
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein # p v- z2 H9 {8 RInteractions (RFAP) in Live Bacterial Cells & ?& C% @0 e: E# i+ ]20. Chapter 18 Cellular Aging and Death [, J- T6 a# D9 {
1. Introduction " p( H4 B; W4 b2 g4 v) [2. Unit 18.1 Current Concepts in Cell Death 0 _1 v1 I+ c# H ~+ O7 S3. Unit 18.2 Analysis of Caspase Activation During Apoptosis 9 c* |" C. x$ h/ s4 B t4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria , e1 d: B. Z% x k v! p5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH), h: s( W/ g: }6 B8 H
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death + d, h+ s- q% {' N! Y4 m7. Unit 18.6 Analysis of Telomeres and Telomerase ( N6 A* t! T0 m8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of& \6 z+ e+ [/ r" ~! U# }1 _
Poly(ADP-Ribose) Polymerase ! W7 p6 R0 ^( A% X5 v3 _% c$ p: b9. Unit 18.8 Flow Cytometry of Apoptosis n8 A" x' Y: J6 |' k" M
10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase ; i5 O" R3 Q: `( SAssay 8 Q& a" Y2 G* Q7 i H) u11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis * m) B( c a0 R21. Chapter 19 Whole Organism and Tissue Analysis1 J6 {$ K0 ^3 p* P. N/ s
1. Introduction9 s, S) c6 R7 X; I
2. Unit 19.1 Overview of Metastasis Assays2 z( b6 g) W& A4 G; v5 [
3. Unit 19.2 Tail Vein Assay of Cancer Metastasis' Y" p) y& g6 F7 E4 r
4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene) J! W# e4 Z" S0 Z+ N
Expression After High-Fidelity T7-Based RNA Amplification ! h$ A% F5 Q3 T: Y1 \& C5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA8 _( ~! n9 C O/ J2 c ~4 M8 B- R6 u1 `
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model! j' U# m# }' Q; `
7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo. ?8 B0 H, T/ B% e
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo ! w' a4 R, ] F8 O V6 Y9. Unit 19.8 Embryonic Organ Culture 3 ]; R. ?* S& h, C10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin/ ^, S8 O8 f( _2 ]) c! J
11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice , ]$ H4 _" I' v ?9 T# I/ P o# t12. Unit 19.11 Generation of Transgenic Mice: s, S0 V. p$ h; f
13. Unit 19.12 Overview: Generation of Gene Knockout Mice % l0 F, ~5 T/ q4 p* f1 z4 |- u+ r' ^$ j) L7 X14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production9 J/ G3 s5 z2 k6 a# P6 E
15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection+ c" h5 V. h F: A. x
22. Chapter 20 Expression and Introduction of Macromolecules into Cells1 g& a; {% K8 `5 u# `0 ]" U
1. Introduction# Y+ @8 W1 Q+ `, N# {5 f$ g* M6 D
2. Unit 20.1 Direct Introduction of Molecules into Cells . L: h- r% u( _+ j3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System& l' X" E( p6 W+ R7 C; ~0 L& r7 j
4. Unit 20.3 Calcium Phosphate Transfection 5 D0 K" H5 q! [5. Unit 20.4 Transfection Using DEAE-Dextran- U2 M! n( W5 j+ d7 @1 t
6. Unit 20.5 Transfection by Electroporation , C# j6 k9 l4 F6 o0 s, V7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents : z" ^+ ~$ {& x. u8. Unit 20.7 Optimization of Transfection$ T9 J" V- z9 O6 T, d
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System ! N5 B4 v+ x* S! k23. Chapter 21 Fluorescent Protein Technology& Z1 [# r, M* v: N# x
1. Introduction/ Z" M+ \9 t& N: e
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells- L! q2 f* j; H, ^* p
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP) & m- l8 @' A0 c4 v/ ~2 e4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence 8 N3 U' E3 a( D4 kComplementation (BiFC) Analysis 1 m$ L( N. p6 @# M5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology 6 D3 t. D. |- m' g8 k7 K9 B4 Y1 n6. Unit 21.5 The Fluorescent Protein Color Palette4 L. a: T2 ?* t7 k
7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins 5 p. C8 i; w4 t/ v. @4 A24. Chapter 22 Cell Biology of Chromosomes and Nuclei & C; D7 P; P8 p) c! S' {! r1. Introduction0 \8 q" t/ j& L6 F; n
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis & ]$ H8 {% ^4 L ?' T3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples 1 Q+ I' l) e# D' p. n* y1 }4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis7 W' c, h- e+ G c" X, k
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)% Y7 _, b" X9 ^. q: g; q$ p
6. Unit 22.5 Multi-Color FISH Techniques 9 X; Y& ]* y# J7. Unit 22.6 Comparative Genomic Hybridization. G6 _3 X( A7 K$ v* A# H7 ^0 A4 {
8. Unit 22.7 Sister Chromatid Exchange - q7 `; m W4 O. i( B9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery: m, X7 X! j7 g3 u
10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes , d- l: s( n3 Q$ _# Q4 _% k11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs 1 J# i0 G0 }- X. Y) q& F- e12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly# [6 Z# V* S3 x G2 n8 i d& \+ U- ~* C
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells1 m: C) {7 M G# S
14. Unit 22.13 Monitoring mRNA Export% x3 f; Q- A$ o1 Z6 w1 O! C2 Q4 Q
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-: c K* R* B) c& T% @* a
Field Gel Electrophoresis+ k* G B6 w U2 W7 N9 I# D) E
25. Chapter 23 Stem Cells $ _5 h% \6 h7 p1 c% t! D1. Introduction! v+ E4 s6 D `$ \8 C
2. Unit 23.1 Stem Cells: An Overview 9 w/ P% A& t6 {3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture6 C' q% a$ j' R E( U. ^
and Differentiation as Embryoid Bodies i, B* O$ r( m& \4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood & m h& ^' v$ x- }Vessels0 D, k; B; q9 y% |# o' I4 N m
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into* Q2 d1 g0 |& J, r; X
Adipocytes9 c3 Z& ^. z2 ?6 u# @
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation- C) b) i' k" T( h
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells8 b8 q9 N) a% E/ Y5 @2 f
8. Unit 23.7 Neural Differentiation of Human ES Cells% g) n+ s1 C9 j7 V3 r2 H$ s4 o
26. Chapter 24 Lipids, n6 b& ^$ t" E
1. Introduction 7 t1 n+ f+ p2 b6 J, a& L* H2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking + H ^8 h- l1 _' D* p' e2 u2 |3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins 9 Y" ?$ v0 W" ~1 C: y \2 ?4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film 3 U+ L" `/ v1 |9 P7 C2 X6 v3 n5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains ' T7 }' Y: T e; }0 q27. Chapter 25 Nanotechnology 1 r9 L5 q0 D7 V3 G( {3 i1. Introduction! l1 t: o6 }1 {/ C
2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes* a- i3 q4 P# W( ^
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering % R9 E2 J% `: g28. Chapter 26 Viruses! \) r0 A* p' D5 u0 p6 g
1. Introduction ; P" p0 U. A5 K9 f1 x) `2 Q2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors$ V: t7 M; g) F$ m# G, S
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of. x8 V8 I7 @6 r7 D9 b
Cell Entry# s: h. c6 f- K( V" m! g4 t
4. Unit 26.3 Methods Used to Study Respiratory Virus Infection& L) k, X% O; B+ a7 p5 n6 d
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses 9 S0 B( }" Y0 i; F; k% ]6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission5 x" k3 j' D% G6 v/ g$ B" D
29. Chapter 26 Lipids# l8 ?1 f6 O: A% c$ n& S+ h
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by! K+ y. s0 X4 g& N
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication) j) I$ e# q% D2 n; p$ J9 ~
30. Chapter 27 RNA-Based Methods in Cell Biology+ t3 L5 H) b; f& @0 B; k$ V
1. Introduction& E; C; F Q- \7 M) i5 k9 j* P$ E
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs 6 r" |! W; w* J" v" `3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible5 t: Z; {3 O+ G7 U! T! e
Rescue in Mammalian Cells 2 o$ W/ M2 G7 P( |0 \6 ~3 V$ G31. Appendix 1 Useful Information and Data - a4 I- b! Q+ ?% k% ]1 @1. 1A Useful Measurements and Data 6 \7 k3 B! \( G: j2. 1B Compendium of Drugs Commonly Used in Cell Biology Research $ V% k& @2 y( Y# z4 H4 d0 K5 ]3. 1C Identification of Motifs in Protein Sequences . u1 J" _0 l3 n3 K0 W4. 1D Safe Use of Radioisotopes 0 X2 b; {) v9 V! B5. 1E Absorption and Emission Maxima for Common Fluorophores : T1 B# |( L o1 r9 n/ { j# Y6. 1F Importing Biological Materials8 l& e" j6 g$ n6 `( }
7. 1G Centrifuges and Rotors% D! i$ j0 _: F1 e! b1 L6 G
8. 1H Internet Basics for Biologists. R# ]0 L- U1 u9 P
32. Appendix 2 Laboratory Stock Solutions and Equipment ) R2 q8 O r1 M0 x, ]! Z1. 2A Common Stock Solutions, Buffers, and Media & c* x, V8 n* p9 }2. 2B Medium Formulations , G) @2 s! m2 ]+ Q( f0 \3. 2C Standard Laboratory Equipment" B! O! q S& i. b; c
33. Appendix 3 Commonly Used Techniques: V; |# r2 t; A. L3 J8 n+ P
1. 3A Molecular Biology Techniques 1 a$ k, N4 Q0 O" _7 k2. 3B Spectrophotometric Determination of Protein Concentration * A& n# X6 s( ~* L4 H3 t y3. 3C Dialysis and Concentration of Protein Solutions ! o0 j, L: b) U/ y4 v* v4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy + h9 w4 ?4 i7 K4 ?- Z" t5 i; J5. 3E Silanizing Glassware 5 ], Z5 l' N6 m) z# X( l$ @6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization3 f& ~2 e3 @: R# t
7. 3G Micro RT-PCR9 E- _7 l# L$ g. T5 M
8. 3H The Colorimetric Detection and Quantitation of Total Protein4 g% Y0 z' x1 a% a( P
34. Appendix Suppliers ! B) V- G3 {" m( F! I- x1. Selected Suppliers of Reagents and Equipment; |( H, L9 m5 K7 T, J7 o
9 J5 z9 ?& ~( d. Y% r# B; A/ c k" i作者: bulu123 时间: 2011-3-8 18:58
