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本帖最后由 细胞海洋 于 2013-1-24 14:01 编辑
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1 L9 i3 ]& t7 u/ LCurrent Protocols in Cell Biology 2010年完整版 5483页
4 G3 O& Q9 g0 B! n. Z$ d9 h1 C
v0 t5 w! y( H# _Online ISBN: 97804711430311 p; F' n7 z0 g' v7 ^7 Y
DOI: 10.1002/0471143030
7 R; e, K& n: y0 y' k7 K+ @2 ^. ?# f5 z' B" K0 W! g7 B
Table of Contents# m! Q4 w7 C, k& G
1. Preface
: Y$ C. _3 R4 t* E8 M2. Foreword( A7 F. E/ ^# k, G) {
3. Chapter 1 Cell Culture9 j( j( b& H" Z1 |: O$ q: F/ h0 ?
1. Introduction
2 |/ `& }6 Y8 F2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture
; F3 P8 v; ]7 s' X" @3. Unit 1.2 Media for Culture of Mammalian Cells; D$ G* d" f+ m$ j9 H3 L" y
4. Unit 1.3 Aseptic Technique for Cell Culture# W& t' }5 L* M
5. Unit 1.4 Sterilization and Filtration
+ o+ l3 E' T8 Y$ U6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
& P. l8 [ i) Q- r4 r3 s J7. Unit 1.6 Media and Culture of Yeast2 J0 W6 e& d* \3 f* c( w! b6 Y/ [
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
3 b9 l/ z; Q3 e' D0 J' N, M4. Chapter 2 Preparation and Isolation of Cells
5 p% l0 M$ `4 ], M# V1. Introduction
& M/ K# H" M' M% R6 V- C2. Unit 2.1 Establishment of Fibroblast Cultures) J2 e, q( T( d. l
3. Unit 2.2 Preparation and Culture of Human Lymphocytes( |& N) S9 M/ \$ k0 C# n
4. Unit 2.3 Preparation of Endothelial Cells: i( R7 j) ^- b( z6 U3 p
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation: k% {- E9 n/ y5 X; u$ s: |
6. Unit 2.5 Laser Capture Microdissection
7 ~# [5 ?4 v2 L9 l" N% \7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures
2 _/ o! s" L, U x8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells& P+ M" V4 f" o: h7 R" {
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles5 s3 Q7 P* d! v. r$ u* K- g3 E- Q
1. Introduction7 N* S2 l9 x. ]# f/ D y8 e7 U
2. Introduction
% j0 K; Y" ^" W- o' G% Q: {3. Unit 3.1 Overview of Cell Fractionation/ e) P( o9 g/ M, w2 Z
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
+ i5 u( h& S# w# h8 H9 X! F5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation" Q. A0 n; K+ |2 }; N) c3 ^: L
6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation
/ H, s! N: \! Y$ x7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient7 V; l7 s9 s. G& k, N
Centrifugation# N8 Z. x5 m# r! L
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient* C- ~5 W! k) q1 S" g0 m: Q
Centrifugation
, S. N' [) i4 v* L9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae( ?% z7 Q2 M( ?" i; c& P1 M
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
3 |2 |$ _8 ]1 ]; a4 e, c11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient# ?: T7 Z) T, K. S* k2 i, i* U
Centrifugation/ S* s/ `& m5 A
12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
4 b z6 N+ H$ H) Y" H, U13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes6 z8 I6 i$ ^5 k7 u& Q6 B
14. Unit 3.12 Isolation of Synaptic Vesicles
8 g9 A. y3 E! H5 Y f' Q( n15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation, N( M5 O$ ^+ d- R
16. Unit 3.14 Isolation of Melanosomes7 ^; U! m7 V; d
17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation4 ^) h0 O' N! a! X2 h% `0 H1 K
18. Unit 3.16 Isolation of Mast Cell Granules
7 m/ R4 q+ k+ E% M9 A19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
; U; m7 t/ ~' R. B- {20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas9 ~* C6 @' j- A
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons
$ U. `0 g0 a0 d: E$ J* m( C' j$ {+ B22. Unit 3.20 Isolation of GLUT4 Storage Vesicles
% a+ ]% T& ?" @3 y! {& {4 |23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes6 {/ k4 c& T* x+ S# _! S" w
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological* K8 w& C, m0 [! Y" h! D
Fluids0 ?# B! @$ i; v4 @' ^ m( p `2 m3 m
25. Unit 3.23 Isolation of Intermediate Filaments* K3 x* H q) P9 d9 h9 I7 }/ `7 L4 @
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle7 J) y8 b/ E% J" }8 Q/ S/ P& ~
27. Unit 3.25 Isolation of Myelin
! k, w. L" ~( B' j! I28. Unit 3.26 Isolation of Renal Brush Borders1 l- e: S/ ~0 m( F1 F) {& ]
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane% s+ A: I6 H+ P% J" h" s
Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts
2 l$ e( q2 s, t! }) F, @30. Unit 3.28 Isolation of Amyloplasts( N- _- V) E' Z+ F
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins
0 n% ?, K* w4 V8 l9 m$ O32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
2 x5 Q4 @5 C3 o$ j" X$ D2 m% XCentrifugation9 }4 J9 S# w: W: I# W0 b
33. Unit 3.31 Isolation of Neuromelanin Granules3 T8 U/ N0 X) f& G$ B( G( Z: l
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and {- c8 x, C. f2 k% l" x
Density Gradient Centrifugation
8 J. B" v- X2 |8 x! `6 M35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
; Z' j: o2 I5 c& g. i& I36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation
+ N) n8 f3 r2 A: I) n37. Unit 3.35 Isolation of Platelet Granules
1 |% H) Q1 V6 L9 g7 E, m" C8 `38. Unit 3.36 Isolation of Nucleoli
) d5 m7 c; }& c39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins6 S) {3 t' n% @
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
/ }; V! V2 W1 c. q41. Unit 3.39 Isolation of Chromaffin Granules
( k$ L- q8 h- t2 B9 c1 q# @42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
) Z1 m: k" f+ [+ ?6 U9 T6. Chapter 4 Microscopy
6 }, V( U+ J9 u% S, D1. Introduction
: j, I# J' G( B4 i2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
+ `+ D) @9 d' P B! Z3. Unit 4.2 Fluorescence Microscopy
$ B" e4 j7 I2 {4. Unit 4.3 Immunofluorescence Staining3 b# b8 e! z/ P8 N
5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
" y) m+ M9 r" e0 |! z! D6 Q" A6. Unit 4.5 Basic Confocal Microscopy; Z0 f. P$ p& ]% F3 G
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues
) [( P+ o' d+ v0 P0 ?- [8. Unit 4.7 Cryo-Immunogold Electron Microscopy. C5 D8 ~% X4 o! V4 `) q
9. Unit 4.8 Correlative Video Light/Electron Microscopy
# W' L4 s! D' k- X' C1 X10. Unit 4.9 Polarization Microscopy9 ~' O! i4 }( t5 f8 S1 }
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
" a% d( x: ^' w$ x4 U: Y/ _1 x12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues. {$ T; H" ]8 z) Y# I
13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface
& k! o+ A5 Y+ _& L' M$ fEvents
( d' w6 j4 B+ |! h4 G0 y14. Unit 4.13 Fluorescent Labeling of Yeast( @ s. ]9 b0 V! W% r; K9 I. ^
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
# n" |% k# R. A9 G16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy P0 K+ W8 B7 v2 V2 g7 J
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules: X* L0 |# f4 a- D9 V
in Endocytosis
3 Y6 ]3 j% r- z5 W, w) u7 i0 B18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
# P* ?+ G' y; q19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development
, i8 G* o. b" n( p, `- O1 T6 }20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images1 T+ L2 ^/ a4 Q7 b, w) r
21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions9 |, e3 W9 K- t' Q0 ?. j
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes5 S7 p! [4 X2 Y
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
8 i' d& u* H& E24. Unit 4.23 Interference Reflection Microscopy5 p3 D* _( F6 J7 K
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach
& k6 M2 ^4 n$ [6 q* b6 k# d26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
& p; T1 i5 A4 \( y z27. Unit 4A Organelle Atlas: Appendix to Chapter 4# r. P1 x, ?4 q
7. Chapter 5 Characterization of Cellular Proteins
, _( M$ y" F1 x1. Introduction
& y# W0 @; b" j) O2. Unit 5.1 Overview of the Physical State of Proteins Within Cells
6 y0 W4 y+ V+ `- p# u; a3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
* _* B2 T1 l& [8 l) D4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients3 p$ v) S! W% y- P6 m; I
5. Unit 5.4 Analysis of the Association of Proteins with Membranes
, x& ~$ v: r2 Q6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)
+ I' c# |, D* i9 u2 K7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
+ S. P1 ?& ]3 q* r) |5 }Spectrometry: h3 T. g/ S! D1 p9 I
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
5 D# M% w7 u. I) [) f3 s0 gApplications u) T% P4 r! X$ K
8. Chapter 6 Electrophoresis and Immunoblotting
. k6 k: { }8 K8 \* d' v1. Introduction
& M$ A# a3 G6 u7 M$ W2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
, o, B8 I) K/ g0 k, Q2 T3. Unit 6.2 Immunoblotting and Immunodetection
( Q8 O0 Y. O9 ]) l: u4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots6 O! h% k4 D0 w1 |+ O/ u5 n& ]
5. Unit 6.4 Two-Dimensional Gel Electrophoresis
. S- b5 G/ m+ w0 T# c) U6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions
* x1 V+ S6 @$ @. y7. Unit 6.6 Staining Proteins in Gels
3 C+ N6 x0 |! c# u0 }3 w8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
8 u ]# M% m2 H; N9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots8 }& O8 z% ~6 [6 L! Z* }/ q! U
10. Unit 6.9 Digital Electrophoresis Analysis
9 z/ L+ [) z. n2 p+ f* j$ y! N11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis
4 G' P( S; w: j, ~- t; H- O12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of0 c4 b( C* h- ?' n! B, ?
Single Cell Gel Electrophoresis (Comet Assay)
5 e0 M$ b* |% X& Z0 ?( h9. Chapter 7 Protein Labeling and Immunoprecipitation4 M! [* U k8 Y, L q
1. Introduction1 x3 A' S9 l5 O! T) c0 h. P/ }- j
2. Unit 7.1 Metabolic Labeling with Amino Acids
7 {& Q, W4 B8 S; u5 Y1 m" J/ J3 `3. Unit 7.2 Immunoprecipitation
; _2 f$ D' ^! m4. Unit 7.3 Metabolic Labeling with Sulfate$ _, ~1 s: U% L5 h9 C/ j6 K
5. Unit 7.4 Metabolic Labeling with Fatty Acids4 \& _9 Q% t! r% T
6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups# O( e5 ]1 a( Y' B
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins
- o! }% V$ q1 f% L4 R- m2 l5 l8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
; w4 E3 D& K- ^' s5 B1 `9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars+ l1 m- H2 z9 p
10. Unit 7.9 Analysis of Oxidative Modification of Proteins3 |' T; s& n0 e$ N6 y
11. Unit 7.10 Radioiodination of Cellular Proteins5 t7 I0 @6 L6 k& j9 g
10. Chapter 8 Cell Cycle Analysis- ?, c( P2 n2 z+ W
1. Introduction$ S" Z) \: z+ Y% H* B g4 J
2. Unit 8.1 Overview of the Cell Cycle
/ m3 T; m1 @9 C. A: q% m, p. m) P3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
$ B& j( f/ O$ c& w9 b; r4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle
: X( K! O% `1 _) _* g! ?5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry
8 n0 u1 ?, I$ ?' O, q+ z6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells
- u5 M1 ?4 H% N& i7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry9 H y: J3 k4 e) K9 z9 v
11. Chapter 9 Cell Adhesion) p6 ?8 B V5 g; C
1. Introduction
1 d: h7 L# ?% j; Z4 }2. Unit 9.1 Cell-Substrate Adhesion Assays# @: u0 ^; o; ]6 F
3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force$ n( I" K9 d5 v
4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
) f$ s( o7 ~1 M2 G5 V9 n ]5. Unit 9.4 Analyzing Integrin-Dependent Adhesion+ q: E0 X' K3 C, A b+ m
6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules5 |4 Y% m( J7 ^5 l6 Y5 I
7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
+ } H U* H9 X$ W12. Chapter 10 Extracellular Matrix p8 L9 Z) m) G* N6 d: d
1. Introduction/ j8 P- }7 {0 r, L
2. Unit 10.1 Overview of Extracellular Matrix
- @5 m/ r" H) r% n) `& A4 p' L3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors
' e2 l6 e+ l \4 x* }4 V6 W4. Unit 10.3 Preparation of Gelled Substrates
0 o. }. v+ G" t I3 K$ S5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9
: g6 I. H$ I: L; S- e0 G6 lEndodermal Cells
@8 s2 Z/ a8 Z' P* p& Q6. Unit 10.5 Purification of Fibronectin
m& \, u( Y( v9 m6 I7. Unit 10.6 Purification of Vitronectin
1 ?! e! A4 A3 c! d8. Unit 10.7 Proteoglycan Isolation and Analysis
% C+ {# Y$ G, U& @' J9 L7 U9. Unit 10.8 Matrix Metalloproteinases) D- {& D. v$ j) e4 P
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts5 ~6 K1 {& l5 Z r
11. Unit 10.10 Purification and Analysis of Thrombospondin-1
% G, Z, G4 M* @! A$ B0 m+ H12. Unit 10.11 Purification of SPARC/Osteonectin
. B5 p, w# `1 N9 n: F) E; o% u! o13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
- y( p: V' ?7 c9 A14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly- l1 t2 L0 R3 ?% B
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor+ ]- g3 p8 D" s$ j! ~. S) H
Xenografts
3 \* ?" l0 h% R4 N$ b16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning
! I3 z- Z" Q q* y$ _2 p4 V17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties$ H, ]: x6 G& W/ ~+ ~& {5 X
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D9 B t+ s0 m0 ]: ?- C$ j
Cell Migration
$ W7 K6 F6 \6 a t19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix
; |9 n1 f9 O7 y8 v& u+ M& A& _13. Chapter 11 In Vitro Reconstitution
0 F) R b0 D. y! J1 f; F+ L X1. Introduction% @" D7 n% H+ D/ u! H& {% w
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems, F9 s! f0 l$ i8 }6 \) W
3. Unit 11.2 In Vitro Translation
7 a7 D% a. B* w' ~# ^8 l4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells' {) N3 X# N K) c7 m
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes4 G6 E" W, T4 r$ C% S. b
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication. j3 B2 j0 S" z2 u. v8 c# Q* k
7. Unit 11.6 In Vitro Transcription: x9 ^) Q! z1 L3 ?
8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells3 _( }. P# l$ s# F6 C
9. Unit 11.8 In Vitro Translation Using HeLa Extract' Q( X) ^( P8 T2 W% [. v R9 N
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems9 A( R; l# x% O# U: U
11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts2 A5 |2 ^5 d w( o6 K% c
12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts( x( }* F, x- K8 m
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts/ A6 G$ T6 w) ]. ~( D
14. Unit 11.13 Mitotic Spindle Assembly In Vitro9 z: Y% A7 |( X5 K. }
15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes1 m' k: I: I) o* B" H& L
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
+ \# x" q$ b0 X17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
: S+ @# u1 P' o6 a" z18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts2 S5 `, Y) ~! A1 \
19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells" A* H8 _7 [6 D5 C, d3 O
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import
# f( I4 Z j& ~* X$ r. r14. Chapter 12 Cell Motility
1 A% h4 m g$ ]0 V: X% P% Q1. Introduction+ D' `. t3 F2 _. k# V
2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells
7 M2 a0 N J7 e9 r9 l" k! W3 n- A0 i' {3. Unit 12.2 Invasion Assays
; T- f; f& P6 Q$ y& p6 u4. Unit 12.3 Cell Traction5 y2 _" n# v" y
5. Unit 12.4 Cell Wound Assays( i; [5 U( ^2 a# ?( [1 v
6. Unit 12.5 Dictyostelium Cell Dynamics
$ Y# v6 P# W' X! I+ Q- E" M7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils
8 _1 `* k7 m# X8. Unit 12.7 Actin-Based Motility Assay3 N1 @/ F0 C. R5 }: D+ P' g
9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
) y1 V3 r8 X" s+ s( F, i9 I15. Chapter 13 Organelle Motility8 y; v; e# W, V9 K
1. Introduction
6 {) x" Y! b1 b9 q5 n/ m2. Unit 13.1 Microtubule/Organelle Motility Assays
" F% q+ B1 ?$ c E3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
% X: t0 ], J4 Q/ O" L* O4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP7 f% c2 _1 [7 V+ K9 V
5. Unit 13.4 Movement of Nuclei
& Y; ^4 Y* E6 r. i6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching6 ` w8 g0 h0 g# ~( |: K
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly8 R) u$ N* k( b- V+ [" W. O& H
16. Chapter 14 Signal Transduction: Protein Phosphorylation H' x4 r. K6 V# D( N ?' Q" I
1. Introduction
- e1 b: L1 l4 a5 c. }2. Unit 14.1 Overview of Protein Phosphorylation
9 m* X3 j$ [) K1 h' ~7 ?- _3. Unit 14.2 Immunological Detection of Phosphorylation
5 x7 }4 S+ m& m; h- T$ ?+ U; i4. Unit 14.3 The Detection of MAPK Signaling( ^) }' c9 O8 X4 s3 I
5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
' j2 t3 Y1 D8 X C8 g3 W4 a6. Unit 14.5 Phosphoamino Acid Analysis
+ b: E' }( o1 U$ y; h) y7 ~7. Unit 14.6 Determination of Akt/PKB Signaling
8 g( w5 C5 {' ~8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events' L7 V. \9 P5 K7 ]4 T6 U& ^ [
9. Unit 14.8 Rho GTPase Activation Assays0 L H7 Z( W' S9 b
10. Unit 14.9 In Vitro GEF and GAP Assays
6 T/ H1 O+ q; e7 E11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance( T% `: @/ ~; p- @, }
Energy Transfer (FRET)1 S7 o# p0 Y( z
12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells
3 m0 j" p; Q4 X7 W; J13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells
y3 K! }0 ~0 R0 G- p- D2 o0 W0 U6 T3 p17. Chapter 15 Protein Trafficking V" t0 l$ v1 p ]1 v
1. Introduction
) E" T' @( H: _1 Q4 w+ y9 Q2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways4 O+ l% F8 q6 _
3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking
, C& k* }9 ]+ m3 S& L/ f5 C7 d4. Unit 15.3 Endocytosis: Biochemical Analyses5 X4 D# b) B! [$ N4 z1 d. ~, D
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane
6 O1 z" `: V" b% A6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
) S, @* y _2 l9 E V6 X. j& M3 ]7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum$ T! ]* {# `1 T+ z+ J( s: k4 [
8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation
9 [! q; |2 T/ m# s& ?- ~7 R ^9. Unit 15.8 Analysis of Protein Transport to Lysosomes7 e* P" c+ O, Y8 Z8 l
10. Unit 15.9 Studies of the Ubiquitin Proteasome System+ H( Z2 L$ Y9 x1 j0 G7 o/ o9 D
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network
. n% M# i+ T. I( P% t8 O2 I12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules
8 o5 F4 |1 G" g* q, @ R" @13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells' c& c8 @: Z9 B* C
14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging4 n% z. @' v& U
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
0 k7 s; D) r/ f2 y' j/ z/ SEGF Receptor
6 A8 z g( K/ O7 d- P16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers
: G' o% e A3 l4 z) s2 [: ^! Z18. Chapter 16 Antibodies as Cell Biological Tools2 H" c# D: i% P3 k# }( J
1. Introduction& O+ ]; z3 p7 c) [
2. Unit 16.1 Production of Monoclonal Antibodies4 P. r3 T3 b8 O) k
3. Unit 16.2 Production of Polyclonal Antisera( {! n4 u8 x7 l' v; |
4. Unit 16.3 Purification of Immunoglobulin G1 E1 z% e( O. V f \; O% J
5. Unit 16.4 Fragmentation of Immunoglobulin G
6 f2 l% n! P# t6. Unit 16.5 Antibody Conjugates for Cell Biology7 u0 C4 N3 G! \0 X: p) y* v$ a
7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides
) z1 b# U* H6 `: O19. Chapter 17 Macromolecular Interactions in Cells2 Y. j) J+ Q7 j$ C3 i
1. Introduction
" ^ w, c- l% f1 a2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)1 N m1 t. Q) @" a
Microscopy; b' V2 P* X+ V5 c( S
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
# `* V9 m# F t2 T3 `4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
# K9 g6 N' m0 Z2 y5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries( Q# y* K5 C0 m9 L N
6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry
* F# [" N8 ~) |+ I- \4 r7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors% h) c; v9 V3 B# }0 K+ D1 O
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific1 e% f( P& j. t! M- t8 c0 Y* E8 ?
Genomic Sequences In Vivo
n4 e( q8 Y% a- r9. Unit 17.8 Isothermal Titration Calorimetry/ @& ?9 L. V/ V+ z/ N* o9 h
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells4 O% H" X, X2 w6 R+ s( d8 m0 @
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking/ t7 h# W- \3 j8 i/ J* j' K) W
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein% J) Q6 d2 K7 {: P4 _& n; D7 u. U4 z
Interactions (RFAP) in Live Bacterial Cells
* @0 s# r3 K. _% d B- z20. Chapter 18 Cellular Aging and Death$ [9 L. y$ O" J: }
1. Introduction
" e4 h, f8 q7 E8 @3 j" o2. Unit 18.1 Current Concepts in Cell Death
; Y, m' E% w" e! _3. Unit 18.2 Analysis of Caspase Activation During Apoptosis# V! Z; F0 |' S+ I% b
4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
: t: g, I# X% X+ m4 ?$ Y+ h5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)% S0 k- u m3 k; t
6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death
: o5 u- B$ `2 L9 X7. Unit 18.6 Analysis of Telomeres and Telomerase+ l. ?( H$ A) U" |2 X' Q" R
8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
6 q/ ?$ u2 a, s T zPoly(ADP-Ribose) Polymerase! U2 G. B# v9 A/ `- z
9. Unit 18.8 Flow Cytometry of Apoptosis
- a6 F8 r) _' s& E |" I10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase' O! A0 z9 R6 D( s* }& R
Assay+ j3 N6 `& H6 _$ B7 O3 H
11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis
9 {0 ]0 \( L5 D" P: o' P1 {21. Chapter 19 Whole Organism and Tissue Analysis
& l: p+ P! P2 d; M, D1. Introduction
3 G ]+ x1 r/ p6 s2. Unit 19.1 Overview of Metastasis Assays
) m6 F+ I' f% a3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
0 ]1 i: E# E$ D* Y% V% ~2 V* t0 o4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene/ {3 X! I1 B& S/ J' r
Expression After High-Fidelity T7-Based RNA Amplification6 }& I0 Y$ a+ e# T* S }, Z" \& H
5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA
9 X' r$ d& x( b8 ?& y6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
x& A* L$ T0 _3 }& D. d7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo: O7 N) b/ H4 b
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
" k# Q/ n& i; j! s2 M9. Unit 19.8 Embryonic Organ Culture
1 k* m8 e P0 Y8 f10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
2 n; h0 O" r) @( l4 i" X1 ^' P11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice
# Y7 O5 }; E2 E: f. k, w* n- b) a1 N12. Unit 19.11 Generation of Transgenic Mice1 R2 R0 q: a1 D; S/ }4 y: }
13. Unit 19.12 Overview: Generation of Gene Knockout Mice
) @4 Q2 k1 o' O9 `+ s14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
% Q# L, V- o+ X8 w% V; {& j15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection3 c A' A* Q: _; } i J+ Z
22. Chapter 20 Expression and Introduction of Macromolecules into Cells3 Z! S5 o( D/ V! T" k% j
1. Introduction
1 w/ ~; b; ?) k; x4 K J& i2. Unit 20.1 Direct Introduction of Molecules into Cells0 L6 q. A. p# o! t) @
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
: z( x+ Z" s( c+ S4. Unit 20.3 Calcium Phosphate Transfection, D+ v$ n7 w/ G% {
5. Unit 20.4 Transfection Using DEAE-Dextran
7 d% W H2 I9 E; s8 y! x6. Unit 20.5 Transfection by Electroporation
( w; v1 E# A5 s* }5 N! M5 ]7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
6 j6 g1 b6 M3 C5 R6 W, F) q+ F' x; I8. Unit 20.7 Optimization of Transfection( q, G9 s8 q% d$ Z( t
9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System
5 L! \4 h+ C& c) w9 O- @23. Chapter 21 Fluorescent Protein Technology5 q0 u1 P( \9 U2 |9 k& j
1. Introduction
# ~6 W- e) M( r3 m2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells3 \* ^0 r$ u4 R; H
3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)
% Z3 i8 d/ z( F# ^8 F X% C4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
/ W" q$ ]6 {* ^( R3 aComplementation (BiFC) Analysis
/ d3 O" p# l! [! |9 A* ?5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology
1 s2 e" x) `0 h) S6. Unit 21.5 The Fluorescent Protein Color Palette
! s! _) q/ |; d6 `7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
2 _' G/ Z6 f4 b Q24. Chapter 22 Cell Biology of Chromosomes and Nuclei0 V4 X8 t% A( P2 G& b) d& N: l6 l
1. Introduction
/ u% \# y. v6 y. z' ?2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis/ J# T) R0 I1 Y: [) Q: q
3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples' ^5 ^- A# V/ b! S9 D9 s
4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis
' Q2 o# P M8 T, a/ E. e! l5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)
2 s4 e$ |) x% r6. Unit 22.5 Multi-Color FISH Techniques
3 r. E, [( c& \$ [8 R4 M# O. U7. Unit 22.6 Comparative Genomic Hybridization" n/ D& j% d) V) h* v; C! M) l+ ?
8. Unit 22.7 Sister Chromatid Exchange8 X3 B! h% X. j
9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
5 ^ }8 q2 }- |& I10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes5 }& m5 _( _; C1 q
11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs
2 B; l5 V5 t4 W12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly8 L! ~' }4 n" M
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
2 f: w" _2 p# a5 y! O: @) C14. Unit 22.13 Monitoring mRNA Export! D0 p" N! _' D: w: h3 T
15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed- V. {$ C4 X* ^
Field Gel Electrophoresis, R5 h ^! n" s
25. Chapter 23 Stem Cells
0 _% i/ F4 J' e3 o/ r) c1. Introduction
4 r9 {0 R( L* y2. Unit 23.1 Stem Cells: An Overview( L. t# b6 F2 Y* {6 d* k
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture- m2 M9 G1 v- B) G: t9 A# O8 E
and Differentiation as Embryoid Bodies$ {# P# [1 l/ ]4 o: c; c! n
4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
3 c! w: b+ w" b( x8 n. M3 gVessels1 s% X" R+ F' Q; C
5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into
' V- H6 T7 j; o' s7 I6 vAdipocytes
# e) v D5 ~9 p8 P6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation
- I( b6 s6 T$ Q& \' v% L7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells! p5 U. Q- H9 F |8 K4 _
8. Unit 23.7 Neural Differentiation of Human ES Cells2 {# _! a- `* i& p/ p6 v: d
26. Chapter 24 Lipids: B+ s0 i; l) }/ v
1. Introduction& f& }% R' @, X$ z4 a3 l v
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking* |9 Y7 w `- d5 `0 q- r/ c* q
3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins" J5 D: J! q/ [1 b. f0 b: i
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film" h0 W9 b! Y/ \7 h' F# r
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains
, C. j Y% ~, Z7 j; U- U" I27. Chapter 25 Nanotechnology# e% V) F9 _/ U
1. Introduction
: c+ G4 A, j- t/ k2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes
; \* F( F% w4 Z+ M: N L3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering
2 \0 F9 j4 L6 N: ^, v28. Chapter 26 Viruses
% y! l7 `( d/ B" v7 d+ i% S1. Introduction
5 Z: n( s" P i8 v/ E0 {2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors% s5 C& Q7 x1 o) v a1 B; u4 K
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of0 i- U6 v& y! d. o
Cell Entry
2 m" i' Q0 T$ S4. Unit 26.3 Methods Used to Study Respiratory Virus Infection8 h7 f% G. U+ G5 W& j- j- `& n
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses
; J. J2 J* d: Q9 e6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission
+ U* H1 C7 i% w7 k29. Chapter 26 Lipids9 a6 e6 [% C- N7 s
1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by) k+ ?$ M2 a# w" m
Real-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication8 B' M, }2 M# C- b
30. Chapter 27 RNA-Based Methods in Cell Biology
0 \( N7 I) C8 A1. Introduction
/ A* Y1 L3 ]. k. q( J6 }) {. N2 o2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
4 B. l q# n8 F5 [- o6 s+ i3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
" l( w" I" X- `6 m/ {7 ?- d- ERescue in Mammalian Cells
2 K8 A8 c) W% G31. Appendix 1 Useful Information and Data
$ t$ |" L/ i$ f3 Q' N# t P: |3 `1. 1A Useful Measurements and Data; {# s, S# M& W$ @! W
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research0 }, k$ U( W7 b7 g% Q% G
3. 1C Identification of Motifs in Protein Sequences0 s L' W$ ?. Z, p% w+ U% K5 Y
4. 1D Safe Use of Radioisotopes
9 |8 H! \( Q+ X% | i9 e0 {0 R5. 1E Absorption and Emission Maxima for Common Fluorophores
3 H" i, v w4 x: R. n6. 1F Importing Biological Materials4 t! I- b8 c2 @/ `
7. 1G Centrifuges and Rotors
- u A$ u$ C ^4 J8. 1H Internet Basics for Biologists
0 t! z8 q6 T) C0 i a$ J1 L0 Q32. Appendix 2 Laboratory Stock Solutions and Equipment: {% Z; n! T9 N0 {1 S
1. 2A Common Stock Solutions, Buffers, and Media
' G! U4 H3 T6 k2 D. `# {2 W2. 2B Medium Formulations8 o8 x9 `8 ]0 l
3. 2C Standard Laboratory Equipment
, d8 L8 r O& k' Z' Z33. Appendix 3 Commonly Used Techniques
" z" b/ v2 S. E1. 3A Molecular Biology Techniques9 I6 g6 i6 n5 Q: i3 S
2. 3B Spectrophotometric Determination of Protein Concentration
- f: z$ F( O$ H1 a8 d* f2 ?3. 3C Dialysis and Concentration of Protein Solutions
' {4 Z$ ]' C; u" g- @; R4 a; m4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
1 D# ^8 t5 h. ^' K; i# a+ R9 {5. 3E Silanizing Glassware. s7 i1 f% g) T7 A7 e/ ^1 p
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
2 V! `; E, \. B7. 3G Micro RT-PCR6 r5 b- _5 c% i
8. 3H The Colorimetric Detection and Quantitation of Total Protein# p/ P& |. G2 u$ n
34. Appendix Suppliers
) z2 c' m' \ j5 T) I1. Selected Suppliers of Reagents and Equipment; i# i0 `8 V" Y2 R
5 b2 f" h& Z0 s6 K" P |
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发表于 2011-3-8 15:53
