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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑 + {+ j- i9 j0 `" w, i
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Current Protocols in Cell Biology 2010年完整版 5483页
* F) X2 ^# ~6 a# T7 Q' ?' d, M% Z2 _2 k0 L( T4 y. |5 b
Online ISBN: 9780471143031; ?9 x7 R+ A" i
DOI: 10.1002/0471143030
$ H7 A) Y% p) C* C" Z1 p! J7 I- D8 J9 M+ \* I8 u) J/ t6 ?
Table of Contents! @: ^. H" b6 ]- [' J; U+ ?
1. Preface( Z3 F# e2 v. Z `1 Z2 t( z
2. Foreword. V' j+ Y: M" B$ h Q! S
3. Chapter 1 Cell Culture
( e5 s* _5 J9 w. D2 \1. Introduction9 V* h# Y3 s- R% y( K
2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
( o) a2 z: J7 H6 c: H+ s) z5 A3. Unit 1.2 Media for Culture of Mammalian Cells
4 S. u" A0 A2 d4. Unit 1.3 Aseptic Technique for Cell Culture6 q' ~) v1 F5 f9 f
5. Unit 1.4 Sterilization and Filtration6 n5 A; N# I* s% x5 A' k n& U9 }
6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
! a. m2 o+ e. D8 S6 K4 C7. Unit 1.6 Media and Culture of Yeast, P; b) j7 \6 r& G/ B3 Z
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging, C) |% |0 ?2 l0 C6 o
4. Chapter 2 Preparation and Isolation of Cells3 O9 J& [. w9 B: ~2 l( Z
1. Introduction$ [: T d% L! N: Z: K$ d! B
2. Unit 2.1 Establishment of Fibroblast Cultures
V# M2 c- k n N4 [7 P: q, [/ f3. Unit 2.2 Preparation and Culture of Human Lymphocytes7 m& h2 M1 L. d f
4. Unit 2.3 Preparation of Endothelial Cells, W& T- H7 H. `$ u3 L6 l6 s1 o
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation
6 y$ @4 a- f% p7 X/ M0 |/ f9 s2 R6. Unit 2.5 Laser Capture Microdissection
5 |% W; j5 W6 X7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
$ v5 g& q0 W" q8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells/ T9 q* N: T1 \6 Z& ~* o& b. b* i
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
$ F4 }. C# O# @! P$ {0 B, R! q4 g1. Introduction, I: l1 f9 J9 m* k
2. Introduction
- z$ h1 l5 J; G! G, r- h/ e3. Unit 3.1 Overview of Cell Fractionation
& l- Z5 D7 D- N8 M( V# m4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
4 t. q5 E& r* Q3 ^8 d$ v. O5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
) b& E: _; m- {# S" ]: v6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
" Q5 o x" g9 t1 T* `) O7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient$ F/ {0 s' R8 ?- R3 Q0 R% B8 ?
Centrifugation" M; o. l' z, A$ o! h9 M
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient% A0 S* t b/ |
Centrifugation
$ q; u$ S) T/ b9 H# E9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae+ d( y( T$ q$ l3 G: D" D. N
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae5 G* S1 [1 ]" ~ ], I$ b
11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient+ O7 E" S; o4 k# q* X
Centrifugation
2 z# e1 @: i* J4 l6 `( \12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
1 H( L4 q$ u& p) @13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes
* s7 _0 g8 s% Y! A- z14. Unit 3.12 Isolation of Synaptic Vesicles# p# J2 G) l. D9 N6 x
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
8 ^% W5 m2 _: A( x- Y" G( b4 _16. Unit 3.14 Isolation of Melanosomes
6 e+ [. k8 x7 p- j17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
& J" A9 I; B" h9 J0 O18. Unit 3.16 Isolation of Mast Cell Granules
( Z) G5 |! M# H$ l: M0 T19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
/ X4 E6 l6 |5 a' r20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas
8 Y2 c$ V- v1 @6 g8 h21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
# g" V- _3 i' A, y. d22. Unit 3.20 Isolation of GLUT4 Storage Vesicles+ d& W$ }) {' _& {# p5 T, {
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes( {; x1 j! q/ D" P- p# d3 `2 t
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological
( |- b/ a- R4 `/ t/ z5 IFluids( `) Y. f; p; V3 h2 G
25. Unit 3.23 Isolation of Intermediate Filaments3 v; I, e2 H0 h Q6 d
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle; T" d6 P" P. ]) C
27. Unit 3.25 Isolation of Myelin
( ?/ Y+ |5 v1 f1 m: u# H28. Unit 3.26 Isolation of Renal Brush Borders
" U8 e. o8 k" J8 O29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane; v" m* A4 Q/ [8 f
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
% p1 M8 b6 p& W( u, U30. Unit 3.28 Isolation of Amyloplasts" s- K# X1 g7 U- R% W
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
2 r" A% b) ~; f+ j; x8 C32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic0 F+ ?0 x4 U( i# L
Centrifugation
2 K, q7 G/ W/ H) d9 q33. Unit 3.31 Isolation of Neuromelanin Granules
1 f& U$ N8 w* Y34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and1 D) ]$ I& M. i, f4 K" m# X
Density Gradient Centrifugation& J0 ~3 I+ T- O7 m
35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
% U, i' Y& X3 }! j# e36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation; m4 x4 Q7 [6 K( j3 t9 |
37. Unit 3.35 Isolation of Platelet Granules) s2 S( I' }2 y0 X
38. Unit 3.36 Isolation of Nucleoli
, A4 i4 V- d! R39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins. R$ C, k) _' c, I8 ?
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
3 k2 p$ h( P5 O6 f9 }0 @41. Unit 3.39 Isolation of Chromaffin Granules
" @7 K2 S R, Q7 |4 n' F) A$ C42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
; c7 M0 o% P/ `$ ~6. Chapter 4 Microscopy
4 ?# p* _' L& i1. Introduction. F, [4 b. T9 z o3 Z+ z
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
1 v' M0 ?1 q! B) W# L+ Q3. Unit 4.2 Fluorescence Microscopy
" ~$ r0 h l/ A4 k4. Unit 4.3 Immunofluorescence Staining
0 B; C; w3 m& t5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
5 c& @2 t4 K b, H9 `6. Unit 4.5 Basic Confocal Microscopy" ]/ c; r" U. a" t7 ]1 Y
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
3 j& w8 ~5 t9 b/ y: Z/ W8 J8. Unit 4.7 Cryo-Immunogold Electron Microscopy: P6 b& x) b* X: v7 d- N# A
9. Unit 4.8 Correlative Video Light/Electron Microscopy
1 Q% S! S$ Z% _/ H10. Unit 4.9 Polarization Microscopy& {5 |8 b! r2 d' H& ~+ N7 {, T2 @3 h
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells2 H Q) \7 b8 d) `+ o9 B) d
12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues# L5 w$ a; H( u; v! `
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface* n( p& E& F! r
Events8 h% T* H2 N4 F0 }- i$ z4 ]7 Q
14. Unit 4.13 Fluorescent Labeling of Yeast
( o+ I5 |5 J# d8 x" R0 R2 p15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
8 t: ]. v! D4 v; s& P16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy
& |1 j, }5 b! w/ w* j3 {, _ r17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules3 m# F( V7 Y3 L1 c: Z3 P
in Endocytosis
1 d' c. b& i S! G% K5 n. W D18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology x f# E8 k8 f$ C, D
19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
0 c$ r) Q- z- c4 H: |9 O$ q; z# x20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images( W3 Q. ?1 T5 J1 |1 W' a# C
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions
, x6 X9 Z: p7 V3 ?22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes9 y' j' V- }0 r$ M
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics# A- ~+ s3 Q6 D, |- j% O
24. Unit 4.23 Interference Reflection Microscopy
) O( P, K0 c& ^: W/ q25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
" Y% Q# F/ R6 v# _( Y# |26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
; f2 H) y. Q. a27. Unit 4A Organelle Atlas: Appendix to Chapter 4
, a! t2 J, ~- g! [6 R6 r$ c7. Chapter 5 Characterization of Cellular Proteins5 [/ ]! }- g) I; X0 D# b9 l
1. Introduction
( D0 ]$ V/ W2 Q8 G- z3 q' Z- \4 s2. Unit 5.1 Overview of the Physical State of Proteins Within Cells+ b3 C7 l9 d4 i/ H( m4 o
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein+ Y! Y8 V8 Y; w2 U
4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients# J9 b! E' Q8 q! p* [- m
5. Unit 5.4 Analysis of the Association of Proteins with Membranes! w! a+ C O6 n3 t
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)& \) F0 y# K; d, N# I8 f: K
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
+ \: p, r) K( k( ESpectrometry
6 m& s6 t+ P6 {6 _& [8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
* b7 `( D9 W( L' RApplications
7 C* e, b3 X9 s. I8. Chapter 6 Electrophoresis and Immunoblotting
. K- A; g# G- n% ?+ k \2 Q% ^' e1. Introduction
5 J9 R4 p5 Q! ?# y, C( Z2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
$ N" B6 v$ u; |) K& j; Z3. Unit 6.2 Immunoblotting and Immunodetection
: B; j7 d1 Z5 K/ z4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots
; `! }; p; I v2 y6 n. M5. Unit 6.4 Two-Dimensional Gel Electrophoresis
3 T3 w( A K4 V* u; D! ]& ?6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions2 J' [/ X0 k6 }5 o( A
7. Unit 6.6 Staining Proteins in Gels
! y& l- L' W- f, y: }' k; o8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
2 S: v/ V, h+ x1 I7 [9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
) ?; Q( a- M9 n10. Unit 6.9 Digital Electrophoresis Analysis7 l. G- q9 I" \; ?, |# n
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
: ~5 \7 B2 h9 w1 q/ ?2 ?& `12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of6 c4 ]/ _, } M0 d2 v. A
Single Cell Gel Electrophoresis (Comet Assay)
0 \0 q! }4 j- {% G1 O7 r' e, J% C9. Chapter 7 Protein Labeling and Immunoprecipitation
0 O8 l. F# Z' d" y1 t& r5 a1. Introduction
; `2 a$ d/ Z3 I8 v* C! g) Y2. Unit 7.1 Metabolic Labeling with Amino Acids
! x9 V0 h# M8 H% |, [5 P3. Unit 7.2 Immunoprecipitation
# f! q V o E8 E7 ]; O4. Unit 7.3 Metabolic Labeling with Sulfate
5 k7 V/ v+ [8 j" t* z1 U5. Unit 7.4 Metabolic Labeling with Fatty Acids
* P8 ?& T( |4 m: Q( W6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups' w; A7 x7 d }- Y3 F
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins8 M1 D$ \; \. Z" O$ }4 Y4 [
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins( c4 D- w$ d6 C9 N9 ]- d
9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars$ U) n3 n9 W2 D! |. o5 [1 e
10. Unit 7.9 Analysis of Oxidative Modification of Proteins
% x$ H( W, _9 _# |; n, A9 E3 y% l7 P6 K" j11. Unit 7.10 Radioiodination of Cellular Proteins
' c5 v( _' V6 K* R- b! @0 X6 @. n10. Chapter 8 Cell Cycle Analysis
$ W) {' l& M' q2 S1 W1. Introduction1 S3 J9 t- V! ^8 T2 W
2. Unit 8.1 Overview of the Cell Cycle3 C& e) j& j; r* \
3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle+ J$ a( K- \" w; u) M9 @
4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle! i' c, l) D9 H d
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry; T8 J: z, C, S7 `
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
/ j7 [2 S) ^& l7 W7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry
( ~/ \# v/ m& s3 a p8 m& }: s11. Chapter 9 Cell Adhesion) H. Y- ~ u- n/ E
1. Introduction
3 i1 q$ q& `% _; m3 G" c2. Unit 9.1 Cell-Substrate Adhesion Assays
' ^. {" g! Z9 g( g2 D' o3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
! G2 L; k0 R v% `2 c* o. i4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
/ U( d8 Y7 g/ W5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
/ I% W4 B6 ~8 o* B( Y% B6 \. ]6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules+ Z4 y, l/ ^ z9 _# D8 e: \! R! l- m
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
: J5 j: d2 [0 Z$ V2 W12. Chapter 10 Extracellular Matrix. n% k% L- p, i9 E% _: T0 @
1. Introduction
0 j; B" N5 U: N) `2. Unit 10.1 Overview of Extracellular Matrix
$ s1 i5 e4 {9 R3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors+ g1 Z3 |' D8 v: L$ ]
4. Unit 10.3 Preparation of Gelled Substrates* Z, d1 c: `) ]9 J) }1 n
5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
: L$ j$ p$ q5 a8 J3 zEndodermal Cells, u4 J! W2 i; A3 ]' y# x* C
6. Unit 10.5 Purification of Fibronectin: V0 @. v3 n. j& n+ f
7. Unit 10.6 Purification of Vitronectin
7 y4 t8 A' `/ Z% S8. Unit 10.7 Proteoglycan Isolation and Analysis$ ?- F5 L: e; Y3 o
9. Unit 10.8 Matrix Metalloproteinases
1 T& P- [7 Z1 O3 A- K10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts0 g6 r3 {: Q+ X% X# R
11. Unit 10.10 Purification and Analysis of Thrombospondin-1' v+ R1 R; C$ o& k
12. Unit 10.11 Purification of SPARC/Osteonectin7 Q) n+ u! Z, u* {( u0 ~* A
13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
. r8 e- V& H1 g+ q+ [4 Y! C% n/ ^14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly5 \) Y' G; G6 P6 _5 d$ ` }
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor2 C9 {- R4 B! T" G/ |
Xenografts
) E" H& R8 z1 [# f6 d16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
" M. W- t# E$ q17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties1 D% D0 E) U7 M4 l
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D5 h g6 g. n, |3 u
Cell Migration# T! x2 p3 T- h5 C' Z6 `
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix! x! N' Y( A' X# O0 |; \! P
13. Chapter 11 In Vitro Reconstitution+ z" C& C! e2 c7 S* u! K2 A
1. Introduction
5 F1 P: K! r& X% `; D: L1 T; V2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems M, O. w: S* N( Q. A' k8 @. H$ x
3. Unit 11.2 In Vitro Translation0 D" C8 ]- x, I7 w2 d: l! e
4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells2 @9 Z3 M) Z, {' M3 w- Q
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes
- N) ^0 a# s: ?0 |% r6 d6 {* u1 {6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication9 R* D/ d" T# ?
7. Unit 11.6 In Vitro Transcription
, d( w) w0 L, @( z% _8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells( k4 g: ~9 T* a& m0 D
9. Unit 11.8 In Vitro Translation Using HeLa Extract
) G3 E' N) b$ ^! K' @10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems2 o3 Y: Y& p& b* q
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts6 ]1 v7 z& W+ e7 A' e6 ~
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts
' p i4 D/ K7 I4 a/ u# r7 @13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts! t' J# a- r/ N; k O: _6 l
14. Unit 11.13 Mitotic Spindle Assembly In Vitro
# }* I' }, Y: N- }15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes, {3 ~8 p) _8 L+ O8 T
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
' x' B" ~' r8 C2 e4 U; f6 U17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
/ f+ b7 B C) X4 h: d$ j18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts# l6 s$ T' k s8 H. w2 L
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells
6 ^5 k$ r1 `; n6 T9 a20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
4 W; @+ L- d3 q* T1 `/ Q2 B14. Chapter 12 Cell Motility
5 q; J# ]$ r! Y1. Introduction, ]; d) `& P- U9 O
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
/ K) [ b0 v: ?/ A3. Unit 12.2 Invasion Assays5 k: F4 ~2 V8 p5 a' H
4. Unit 12.3 Cell Traction
& }; a4 J/ b2 f% w& x5. Unit 12.4 Cell Wound Assays1 Q! k3 J8 b/ l$ x6 b* P* a
6. Unit 12.5 Dictyostelium Cell Dynamics9 C# [6 x4 l& @/ `
7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils p! Q5 D! e: X. Q% D7 n
8. Unit 12.7 Actin-Based Motility Assay0 o. u! [+ ^! X9 E6 C1 N% P
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP6 S5 f# n: g0 R1 y
15. Chapter 13 Organelle Motility
; \+ l; f4 C1 H& p: @. }4 J8 L! \1. Introduction
$ ~) _1 x, L' }: {& N( D; I0 w) d) M/ w2. Unit 13.1 Microtubule/Organelle Motility Assays* N$ _6 l! o, q
3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
2 p/ v' t0 X1 T& F7 e4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP
9 O$ P4 ]8 C/ A# B6 l4 M4 x5. Unit 13.4 Movement of Nuclei
* ]( v% |$ d/ U6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching
+ B- X6 o3 Q5 J* b4 U0 j( p" o7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly
+ ^4 r2 E( n0 P2 x K+ A2 S3 a16. Chapter 14 Signal Transduction: Protein Phosphorylation4 b, m2 w; n e [; `4 s
1. Introduction3 |6 U+ F3 v2 c8 z/ p6 J2 g" ?
2. Unit 14.1 Overview of Protein Phosphorylation
# N3 O" Y0 }1 _9 }1 i$ L) H" _3. Unit 14.2 Immunological Detection of Phosphorylation
: z1 B: n' M2 {: A4. Unit 14.3 The Detection of MAPK Signaling
7 C' w" i. R9 `5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
' w0 f( x$ J) n% T/ G6. Unit 14.5 Phosphoamino Acid Analysis' [' `- |7 ?, `) T% ~$ I. z
7. Unit 14.6 Determination of Akt/PKB Signaling* ^9 u- e7 y G& n, P# Q
8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events3 W3 x* N( j# Q& U9 P
9. Unit 14.8 Rho GTPase Activation Assays
7 ?+ X3 q$ K$ _" d10. Unit 14.9 In Vitro GEF and GAP Assays
7 T5 e8 j, H( Y11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
s6 c$ }9 O8 |; M u, o/ xEnergy Transfer (FRET)
* ~. y: i. x c4 c6 q5 C12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
6 X) _. d- w$ L6 r3 ]" r) d13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
: V0 S$ }7 q& m0 z17. Chapter 15 Protein Trafficking
7 ^' m9 w% ^6 B- ?. |. U1. Introduction* `$ C+ j- A/ w- H' p% B2 P, P
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways& Z9 e5 m6 O/ w( [# m0 ~. l
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
4 I" E4 `/ w$ |7 Z }" n4. Unit 15.3 Endocytosis: Biochemical Analyses- e$ J) w" Q! q2 b! Q
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane' O4 A* l& m, E
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
9 w% A; `9 T3 w M# l7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
7 k! w% z; Z# S. |" x8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
, q* g; P% x: F2 [5 v5 C9. Unit 15.8 Analysis of Protein Transport to Lysosomes" T, E) n" h9 Y
10. Unit 15.9 Studies of the Ubiquitin Proteasome System
1 g/ B0 w9 w2 L( _5 n( q2 C11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
( f3 @$ O2 p& i12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules4 L7 O7 G, [3 n0 t6 w! p
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
$ @' W. r' d6 C, F' p ]7 N14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging
3 ~, P# G( T1 T) v8 Z7 n; t, k7 E15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
% q& S" h1 s! o: z' ^8 yEGF Receptor1 |1 P. ?- e. L3 l5 `. i5 \! O
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers/ E) B$ l1 S% Z: O5 Q+ j z9 r
18. Chapter 16 Antibodies as Cell Biological Tools
^; ~3 @2 S7 M& m! c" R$ a1. Introduction
0 A, b6 p- P4 h7 ?. ^2. Unit 16.1 Production of Monoclonal Antibodies
: E* K, W8 d! }9 j9 z5 ]9 s3. Unit 16.2 Production of Polyclonal Antisera* i+ G8 l4 s2 P5 C
4. Unit 16.3 Purification of Immunoglobulin G+ n* y& e ^2 o u& {- T7 {2 X/ e
5. Unit 16.4 Fragmentation of Immunoglobulin G
; R) [8 v3 Z. e+ j0 G6. Unit 16.5 Antibody Conjugates for Cell Biology9 M, F1 D) x$ [0 X' f. g7 \8 q4 H
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides3 R' M" [, N+ h
19. Chapter 17 Macromolecular Interactions in Cells
0 S& _% \: u. M! l1 t1. Introduction
$ U/ Y( \' y# w+ { j: h2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)* L/ [5 N# x" ~) H5 l
Microscopy
' W( ~ }, }/ H/ A3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
3 r9 ]9 Z/ f {4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins7 F# I6 N9 c2 F U
5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
0 M4 i8 x6 V) E7 w. a' F' B6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry R$ [. V' ?; X. S+ S V8 n
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors% ~. } y E' G2 m1 B
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
8 ?& T6 z$ o: gGenomic Sequences In Vivo
0 f; I1 Y7 v: G$ P0 E+ o) e1 W. U9. Unit 17.8 Isothermal Titration Calorimetry6 l0 s+ U1 ~( k
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells6 L7 h; g( f/ Q, [+ t$ P+ K
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking
# F3 M) L6 M9 C# h o7 N- }6 F4 I, H12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
% o. g% d. S* a' u% C$ BInteractions (RFAP) in Live Bacterial Cells
1 J5 [. ^' K, I0 E/ o20. Chapter 18 Cellular Aging and Death7 k1 v) }" u2 V& m' g+ a
1. Introduction
7 A2 o. u" D, W5 \4 R2. Unit 18.1 Current Concepts in Cell Death
3 W7 z) y$ J. e3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
! I' T* k5 p9 J5 j* u4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
3 f* }6 O: o1 D1 n$ z5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
9 Q( N0 ]( i" ?- a, |8 Z6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death3 X. x0 x, ^ ~5 ?6 M7 F
7. Unit 18.6 Analysis of Telomeres and Telomerase
' M. P3 w2 b& O6 o8 j1 T8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of0 t3 @% m5 i+ M
Poly(ADP-Ribose) Polymerase8 l/ a$ W2 `& Z8 A2 c( ~, P) q
9. Unit 18.8 Flow Cytometry of Apoptosis
; A: C5 A6 c! d& d* h- U10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase' w1 {" G* a3 S8 w e. M& }
Assay3 W. B. a% x4 \# K' Y" g: g5 H
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
( T- p7 ^: ]! E9 g1 B5 ~5 x21. Chapter 19 Whole Organism and Tissue Analysis" x8 G0 }! v! a3 J+ Z
1. Introduction
* J" t8 q+ J" z5 s4 J2. Unit 19.1 Overview of Metastasis Assays
( Y" S5 T2 s7 c+ L' q3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
) a3 ^" A/ i7 [4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene& a# X- s% Q' s0 p5 s; u
Expression After High-Fidelity T7-Based RNA Amplification- k! ^+ N, u. a# r+ U
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA4 Q# e7 {* W7 [ ?2 q
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
5 R$ d, w4 q" q0 W7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo
# d5 J( ]2 A- i( G6 Y6 {' e8. Unit 19.7 Imaging Tumor Cell Movement In Vivo) S# x, z3 s j* p
9. Unit 19.8 Embryonic Organ Culture) u, M; u. n* V& X5 h
10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
# E/ N1 H4 b5 e h11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
1 B" m, f* K' a5 ? C1 p' u; r12. Unit 19.11 Generation of Transgenic Mice
! w, t% F2 l) W& \/ J13. Unit 19.12 Overview: Generation of Gene Knockout Mice
5 L5 d3 f; e2 P: K, R7 A14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
% K( V Z7 Q8 |" d15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection
' u; e1 y1 G. o22. Chapter 20 Expression and Introduction of Macromolecules into Cells
, h1 n* _4 E4 |1. Introduction; R+ [( v, F( h8 ?
2. Unit 20.1 Direct Introduction of Molecules into Cells
/ W; ~# H; l1 P1 R" ?, H1 | a+ a3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System! q8 S) z' D- ^; j
4. Unit 20.3 Calcium Phosphate Transfection0 p5 f6 ?: O& V% z6 |9 m i* |$ O
5. Unit 20.4 Transfection Using DEAE-Dextran
e& r4 \, A9 d$ J8 n" S6. Unit 20.5 Transfection by Electroporation6 M1 h- o* {* }1 {6 {
7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
- M( n4 y$ w4 l& W8 A8. Unit 20.7 Optimization of Transfection% z, N% F! N( g' e" R
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System$ G3 V8 d. U% s7 \0 h/ o6 a
23. Chapter 21 Fluorescent Protein Technology3 q8 A7 h* d* a# U2 g2 u5 Z/ t
1. Introduction3 k: t/ m' F' V+ n7 G% J% Q! F% M
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
$ {0 M% m$ ~$ E3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
7 H& w" z/ c) O9 p& Y" U5 V4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
( U2 }" p3 f; @1 wComplementation (BiFC) Analysis' j6 S# |3 P# H; s, e! F; A/ V$ d
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology# `" k. F, S% x3 g1 N
6. Unit 21.5 The Fluorescent Protein Color Palette
- d9 N# A) {4 r' Z0 P1 N1 x7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
9 F# V s6 S d- N) B, M/ U; ~24. Chapter 22 Cell Biology of Chromosomes and Nuclei
0 |7 o* _& I' Z1 S1. Introduction
) N& K3 O: _4 b5 N4 Y: V( b" b2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis, o7 z6 a4 s3 m$ h9 r$ K7 x
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples4 ^9 s; }- g0 O& Y% V, k! q* I, {( Q5 h
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis* P) E, ]! ]& _2 }4 r. I3 y
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH): w) W/ K% P7 ^" j6 a3 c
6. Unit 22.5 Multi-Color FISH Techniques
! X( F3 r( l- Z. X) }7. Unit 22.6 Comparative Genomic Hybridization
/ `: Z2 ^" c7 s8. Unit 22.7 Sister Chromatid Exchange
$ V4 o9 {6 a1 h, w- Q1 m$ E9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
# G( x U. @* Y/ `1 K$ f3 m10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
. o9 S# M5 ]6 V B+ d+ {' K* L- y11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs5 Y3 G/ t; l, V! U% Z$ ~ A
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly) U' B6 Q! x( I# b# d
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
( x, _' m/ d6 ?# e' z/ H8 v# f14. Unit 22.13 Monitoring mRNA Export
% C0 B8 o9 }' z* d2 f15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-! W+ | p$ H6 f# Y% d/ M
Field Gel Electrophoresis& m# s2 E0 {5 ~7 h1 F. i6 U5 v- h
25. Chapter 23 Stem Cells* O+ G( y' R# }4 o! ^6 b' Y
1. Introduction3 Y# M2 F6 b+ h0 l
2. Unit 23.1 Stem Cells: An Overview& R- W+ z) [6 t% b' _4 }( m4 h
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture( ^, g- l% {7 A. v
and Differentiation as Embryoid Bodies, P. C5 c$ i1 K8 z" y4 f
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood8 I. _. h# K( a
Vessels" v+ M V o6 ~: O! _% z, q
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
! f3 o. v. I# k; u+ E4 uAdipocytes
# a, v. w& I: T3 V0 o$ \( U* B6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation% p+ j4 K9 m# t+ {6 \7 S: B
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells
- x: O6 i# G6 {/ t8. Unit 23.7 Neural Differentiation of Human ES Cells
; }" m+ E1 z4 a0 B' i5 q26. Chapter 24 Lipids5 F+ k+ V+ z3 a3 ~: I
1. Introduction3 Z# |1 M$ _' A
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
- X6 A+ ?6 v: w- B0 [/ k' a% b3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins
7 C/ \' v) c# }9 V% h4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film
3 o& l) g* t1 g% p, r8 M3 X7 q5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
0 d( P" N5 y9 v: q7 P. X! g27. Chapter 25 Nanotechnology) A1 r6 r3 y0 J
1. Introduction
1 l/ ?& U# S2 h3 A2 M' _2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes+ ^8 K- d3 m8 S' {$ w
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
0 D( b3 S" z# Y# Y& o28. Chapter 26 Viruses
5 ~ k: ~: g* o _1. Introduction
2 o0 \: n" E9 {( ~2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors( @% A. c. M" @8 b$ _ f( W: p3 X2 V
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
* O2 F: R& `5 z+ ?7 ^9 {- W XCell Entry
7 W" Y: m* [; X2 F4. Unit 26.3 Methods Used to Study Respiratory Virus Infection$ r n) _' B- U" N7 p1 h
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses1 Q3 f5 j- S5 Y% P
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission$ o" L; W/ q0 h. R- N
29. Chapter 26 Lipids
. G3 Y4 D$ S0 y, B, t" u1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by& G+ V4 F& T, p; D w9 h
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication" h/ p) G. G! o
30. Chapter 27 RNA-Based Methods in Cell Biology! r, `5 @. P" _2 _, ~; { [
1. Introduction* ]4 W/ ]! X- ]; t
2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
9 i' X z M$ I3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
1 P# b0 k2 @7 a1 j/ R3 @, H0 {Rescue in Mammalian Cells4 c F( ~- }4 ~/ F3 Q0 a
31. Appendix 1 Useful Information and Data3 h- H# q1 o7 a. w* Z9 {
1. 1A Useful Measurements and Data
8 U' U& f6 ?/ a9 d! d2 [9 [( i2. 1B Compendium of Drugs Commonly Used in Cell Biology Research! e6 r" W/ m3 R& c
3. 1C Identification of Motifs in Protein Sequences# Z1 j7 y! V" v' F% p
4. 1D Safe Use of Radioisotopes
4 U5 V: \5 a4 R' q9 I8 p" ]8 p5. 1E Absorption and Emission Maxima for Common Fluorophores' F+ M9 n' R5 a" B) W
6. 1F Importing Biological Materials
$ b4 o' S' U. @* q7. 1G Centrifuges and Rotors
/ A8 d& y- x m8. 1H Internet Basics for Biologists
& w# ^4 q' i" {32. Appendix 2 Laboratory Stock Solutions and Equipment- O8 t2 t9 E z. `7 ]! ?0 {
1. 2A Common Stock Solutions, Buffers, and Media
5 W J7 k! k( Q2. 2B Medium Formulations
! f. ?3 E4 m+ f0 j* z" {5 q3. 2C Standard Laboratory Equipment
" ^; C9 L/ }1 v8 E. P33. Appendix 3 Commonly Used Techniques
1 w: v, B& R7 ^3 a, S1. 3A Molecular Biology Techniques
3 D! [( F+ x D7 d. h4 M2. 3B Spectrophotometric Determination of Protein Concentration; d" @" a. X6 l9 m
3. 3C Dialysis and Concentration of Protein Solutions
, y( K6 \* ?" A; O2 V# |% \4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy ^% H1 f! S' [ u/ B) r& U, c
5. 3E Silanizing Glassware
3 x9 J0 A7 H7 I6 g5 b9 E6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
$ }9 S T( n k; U+ j4 n7. 3G Micro RT-PCR) p- p; t. l: h+ D" ~) D
8. 3H The Colorimetric Detection and Quantitation of Total Protein9 g! [8 p( }, T" x% ?' n0 J2 l& M
34. Appendix Suppliers
# E9 s3 ^) Z. L. K. p1. Selected Suppliers of Reagents and Equipment6 J0 d/ x& b9 q( {2 W. h% j
% b# w' m s B$ T% G9 w
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发表于 2011-3-8 15:53
