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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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Current Protocols in Cell Biology 2010年完整版 5483页
: h3 x% v! T2 ?; ~* ^
! p9 j) F- s2 u/ HOnline ISBN: 9780471143031( P; N0 r$ ?9 D
DOI: 10.1002/04711430309 \* X% e, \- J4 F$ ^9 ^) D
# N5 f$ g1 q/ t* F7 ?7 j) `! Z$ UTable of Contents
' _5 z9 y/ a& t" E7 n/ y1. Preface+ h/ S( t+ A) \+ r
2. Foreword
- n- f K9 J! x3. Chapter 1 Cell Culture
8 C% H6 I5 P9 Z7 K; O5 m1. Introduction
( A! N( ^: H: k2. Unit 1.1 Basic Techniques in Mammalian Cell Tissue Culture( J+ `# c; @1 U; S8 U# o
3. Unit 1.2 Media for Culture of Mammalian Cells B t2 R r* c# c1 j+ _: Z
4. Unit 1.3 Aseptic Technique for Cell Culture
/ ]6 b+ z3 t2 ~" v# s: N5. Unit 1.4 Sterilization and Filtration
h+ O2 O: {% D! y, A% G6. Unit 1.5 Assessing and Controlling Microbial Contamination in Cell Cultures
3 B7 Z: {6 U- @* H$ \5 K' R7. Unit 1.6 Media and Culture of Yeast& r: L6 x, G- c' S( T8 y. p7 d( d
8. Unit 1.7 BY-2 Cells: Culture and Transformation for Live Cell Imaging
7 o$ T, y0 |* o4. Chapter 2 Preparation and Isolation of Cells; ~) o7 U8 Q% U' Z+ a4 b4 l# M# z6 r
1. Introduction
& r# y, d( s0 o2. Unit 2.1 Establishment of Fibroblast Cultures3 `6 ~- } u$ b0 g/ K
3. Unit 2.2 Preparation and Culture of Human Lymphocytes
1 ~* q1 r0 T0 P# P7 [0 l( Z4. Unit 2.3 Preparation of Endothelial Cells# u1 X4 d3 `& t7 k! D* Z! m
5. Unit 2.4 Generation of Continuously Growing B Cell Lines by Epstein-Barr Virus Transformation9 s( [+ O8 P. w$ j! P
6. Unit 2.5 Laser Capture Microdissection, ~3 }4 J! ?% S& O$ U& R2 a: M8 V
7. Unit 2.6 Preparation of Human Epidermal Keratinocyte Cultures. J/ U! W5 `) [: _3 h3 g
8. Unit 2.7 Preparation and Coculture of Neurons and Glial Cells) g8 |" n( x% O
5. Chapter 3 Subcellular Fractionation and Isolation of Organelles
f+ D( f6 G% N6 }. r' U1. Introduction
$ p y3 p3 r- k5 n$ r* R+ }' r2. Introduction3 X& J" o; @ |9 @& I7 H" l
3. Unit 3.1 Overview of Cell Fractionation. _6 Q- ^/ n: A# V9 B; j6 Z
4. Unit 3.2 Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
) S6 U: \! j) c5. Unit 3.3 Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
6 p! T! ~1 Q% I$ S4 o' z2 Y$ _: o: y6. Unit 3.4 Purification of a Crude Mitochondrial Fraction by Density-Gradient Centrifugation" o. ]# R; g3 `* D: e* m! z6 m
7. Unit 3.5 Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient9 w/ i/ s$ g* G: Y
Centrifugation* j% N5 c: f4 |! A
8. Unit 3.6 Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient
4 `' P' m6 Q7 bCentrifugation8 V. a1 {% ?" I1 f
9. Unit 3.7 Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae. h+ v/ N @4 {7 d9 Z0 N0 M
10. Unit 3.8 Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae
& t$ O* o3 z8 C7 f: l+ T' C11. Unit 3.9 Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient
' x1 n1 K8 P1 J* D! q6 cCentrifugation
9 s4 m. N; W7 K3 J& Q1 _( C7 h12. Unit 3.10 Isolation of Nuclei and Nuclear Membranes From Animal Tissues
: _( \/ S& V/ o4 D: l13. Unit 3.11 Free-Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes9 k! U% t' s, L5 H; Y# B
14. Unit 3.12 Isolation of Synaptic Vesicles: V0 g/ [7 Q2 x: Q: _" E
15. Unit 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation$ S( [% ^% |" J7 C' H/ k: p4 `) v
16. Unit 3.14 Isolation of Melanosomes
6 o$ r6 m |9 u* g. C: m17. Unit 3.15 Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
9 |! R" ?7 k% B. h. |; v2 O* k; @6 L18. Unit 3.16 Isolation of Mast Cell Granules4 b' h4 C5 e* u% `8 z+ `* d
19. Unit 3.17 Immunoisolation of Centrosomes from Drosophila melanogaster
7 C& K7 g- \/ R! u% B. ]3 }4 o20. Unit 3.18 Isolation of Zymogen Granules from Rat Pancreas; h* }) v* \; i9 s/ u4 R6 V
21. Unit 3.19 Isolation of Glyoxysomes from Pumpkin Cotyledons# W. `. l m" R" L1 k2 n( ^
22. Unit 3.20 Isolation of GLUT4 Storage Vesicles0 Q3 u* w: c' m' W
23. Unit 3.21 Isolation of Intestinal Brush-Border Membranes d7 v7 Z1 K- _+ N( d& K8 D$ Q
24. Unit 3.22 Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological) r! ^% b% Z* F) b" Y
Fluids. p/ {# ~1 B6 M4 A! P
25. Unit 3.23 Isolation of Intermediate Filaments" c8 w1 J* n! y* F( F% d- m: p" |
26. Unit 3.24 Isolation of T-Tubules from Skeletal Muscle8 D2 D& q4 l* f8 k5 f
27. Unit 3.25 Isolation of Myelin! ?; q& Y: t$ n8 z$ @: N: Y. Q
28. Unit 3.26 Isolation of Renal Brush Borders0 h& k$ t2 p# j2 T& E& q2 |2 c
29. Unit 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane
; y; j; S& C, D# L- Y& C6 NFractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts4 O3 ~/ q( x2 w3 `: H8 I9 _
30. Unit 3.28 Isolation of Amyloplasts0 ?2 C1 n$ U+ O; p- p! G
31. Unit 3.29 Isolation of Microtubules and Microtubule Proteins/ I. V) d+ J" c1 ]2 u# ~+ c
32. Unit 3.30 Purification of Intact Chloroplasts from Arabidopsis and Spinach Leaves by Isopycnic
$ @* J/ _6 }6 WCentrifugation* r+ o) e |8 |5 x
33. Unit 3.31 Isolation of Neuromelanin Granules4 C( i1 V7 N9 {6 J2 G& N' P k
34. Unit 3.32 Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and5 a, s, [# N) C, x1 z7 t( m
Density Gradient Centrifugation
j( n- @+ S9 f1 ^. `35. Unit 3.33 Isolation and Biochemical Characterization of Amyloid Plaques and Paired Helical Filaments
B# V2 p. J% p, h; d$ o+ F) r5 V36. Unit 3.34 Isolation of Legionella-Containing Vacuoles by Immuno-Magnetic Separation4 v- N, C8 C- d% A" \. y
37. Unit 3.35 Isolation of Platelet Granules' H& _2 _) U) R# K6 F) |( d
38. Unit 3.36 Isolation of Nucleoli, M6 x8 F- x( n4 L7 B0 D
39. Unit 3.37 Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins6 i8 y6 ?; r7 J* M
40. Unit 3.38 Isolation of Aggresomes and Other Large Aggregates
/ t& N6 a% w1 z3 v, ~4 W; b41. Unit 3.39 Isolation of Chromaffin Granules& o4 f% l$ T( {1 I: e" b
42. Unit 3.40 Purification of Ribosomes from Human Cell Lines
2 r4 |) Z. l& c R- W1 t* }6. Chapter 4 Microscopy
2 H6 f% R: T# X% y7 t, w9 |- o4 l1. Introduction/ C, Z) K* G- a- }, }& B1 n
2. Unit 4.1 Proper Alignment and Adjustment of the Light Microscope
) \! c) x# M) ]3. Unit 4.2 Fluorescence Microscopy
H8 Q0 x8 J+ a- {) |" H: J4. Unit 4.3 Immunofluorescence Staining
2 X" u8 x+ X, g+ ^7 G) a5. Unit 4.4 Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria0 V6 `. @$ h% [+ v" O. _) R
6. Unit 4.5 Basic Confocal Microscopy6 e/ j. @5 [, l- A, q
7. Unit 4.6 Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues6 s/ |% K" s/ [" |) }' E! m
8. Unit 4.7 Cryo-Immunogold Electron Microscopy
" V) ?5 E! ~4 y u& |+ D+ ~# @- L0 e9. Unit 4.8 Correlative Video Light/Electron Microscopy
4 i5 |2 V# @% j$ o6 {( y10. Unit 4.9 Polarization Microscopy1 q- N3 f2 @* z% X3 {5 z& Z
11. Unit 4.10 Fluorescent Speckle Microscopy (FSM) of Microtubules and Actin in Living Cells
& S. M: |6 @6 \. f( `12. Unit 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues
' N7 z k* u4 ~" }; ]! }; t9 T7 O' a) Q13. Unit 4.12 Total Internal Reflection Fluorescence Microscopy for High-Resolution Imaging of Cell-Surface3 Y3 y( I% o4 x" R! k* d
Events8 O: F* g/ M0 Q0 b1 o
14. Unit 4.13 Fluorescent Labeling of Yeast/ \0 J$ b/ C+ f$ L( ^# W, D
15. Unit 4.14 Fluorescence Lifetime Imaging Microscopy
9 `5 d3 s* G) Z# n) H# Z+ [16. Unit 4.15 Biological Second and Third Harmonic Generation Microscopy8 ~5 D% _0 t. @& p2 f! h- O
17. Unit 4.16 Analyzing Real-Time Video Microscopy: The Dynamics and Geometry of Vesicles and Tubules2 M* J$ B' b5 q$ l2 o
in Endocytosis
8 O* @, o- ^' H, u4 \3 G18. Unit 4.17 Scanning Electron Microscopy of Cell Surface Morphology
& c- ^3 ?2 `, {( D/ M2 Q' P19. Unit 4.18 Fluorescence Imaging Techniques for Studying Drosophila Embryo Development' m; x7 U+ w- _; u1 d3 u/ c/ O
20. Unit 4.19 Quantitative Colocalization Analysis of Confocal Fluorescence Microscopy Images
/ W+ g& ^2 B1 }4 n. F6 Y21. Unit 4.20 Visualizing Protease Activity in Living Cells: From Two Dimensions to Four Dimensions) P; I4 Z6 Y i1 a0 X
22. Unit 4.21 Photoactivated Localization Microscopy (PALM) of Adhesion Complexes0 V6 s/ ]9 g* l) s, Y' A; a' R
23. Unit 4.22 Culturing MDCK Cells in Three Dimensions for Analyzing Intracellular Dynamics
/ [' {6 x5 j g: P% P7 }0 b. O2 C, k u24. Unit 4.23 Interference Reflection Microscopy# ~% v8 \& W; R3 |
25. Unit 4.24 Fluorescence Correlation Spectroscopy in Living Cells: A Practical Approach V5 v3 X! m' T* |
26. Unit 4.25 Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches
& V* }6 V4 x/ y+ t% U! r+ q/ D27. Unit 4A Organelle Atlas: Appendix to Chapter 4
7 K( z! M* Q$ n2 V v! A7. Chapter 5 Characterization of Cellular Proteins
' B2 X' t7 o7 f" O1. Introduction
# N$ e1 J1 O: ?1 J; g) N2. Unit 5.1 Overview of the Physical State of Proteins Within Cells$ ] m$ X) i P: k' j
3. Unit 5.2 Determining the Topology of an Integral Membrane Protein
, o1 } i6 b! L1 N7 ^* \( A7 O4. Unit 5.3 Determination of Molecular Size by Zonal Sedimentation Analysis on Sucrose Density Gradients; P w/ i, V7 a1 N4 h$ \% W
5. Unit 5.4 Analysis of the Association of Proteins with Membranes# H) p, F7 _" Q2 l; H3 r" o
6. Unit 5.5 Determination of Molecular Size by Size-Exclusion Chromatography (Gel Filtration)8 @( w4 J a8 w2 {1 `, ?0 U
7. Unit 5.6 Identification of Proteins in Complex Mixtures Using Liquid Chromatography and Mass
( k; D4 I3 F+ e2 J) nSpectrometry/ y1 O4 s' b* G) }& h
8. Unit 5.7 Determining Membrane Protein Topologies in Single Cells and High-Throughput Screening
( {' V1 R1 I$ \0 Z3 U: j. u J0 uApplications0 u) Z& R4 b6 u; i! c
8. Chapter 6 Electrophoresis and Immunoblotting7 V. y9 ~- S0 k$ ]' x$ v& H; \1 K
1. Introduction2 N: @* z: c. F% W/ X; j
2. Unit 6.1 One-Dimensional SDS Gel Electrophoresis of Proteins
% H# O- [+ P4 K. B6 C% S" B5 K3. Unit 6.2 Immunoblotting and Immunodetection! D( o9 \' V) x! P8 j& g8 x: j
4. Unit 6.3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots! C* F$ v# B6 h2 f3 ]' `7 R9 K
5. Unit 6.4 Two-Dimensional Gel Electrophoresis" I r) L: Q1 Z/ r+ I
6. Unit 6.5 One-Dimensional Electrophoresis Using Nondenaturing Conditions. p4 z) N8 [7 D9 z/ z) `" v
7. Unit 6.6 Staining Proteins in Gels
9 K- l' W, [, f9 s$ q/ Y1 E+ u8. Unit 6.7 Agarose Gel Electrophoresis of Proteins
* y8 G* J/ P* V9. Unit 6.8 Fluorescence Detection of Glycoproteins in Gels and on Electroblots
9 `8 Q# i: D; F" e4 w+ p+ t10. Unit 6.9 Digital Electrophoresis Analysis e6 Y" P6 N5 q5 n1 t* u, Y! y
11. Unit 6.10 Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis2 t3 `# }: D# Q" l
12. Unit 6.11 Measurement of Oxidatively-Induced Clustered DNA Lesions Using a Novel Adaptation of, M/ @# V! w' R6 U2 L2 L; B; h* i
Single Cell Gel Electrophoresis (Comet Assay)7 m% Q+ b2 M- k% m4 ~; v
9. Chapter 7 Protein Labeling and Immunoprecipitation S y6 c O5 d ^; f) p6 c
1. Introduction# i+ @7 @: [' N
2. Unit 7.1 Metabolic Labeling with Amino Acids$ |4 l: y0 p: ?# ?4 }/ U/ \% v
3. Unit 7.2 Immunoprecipitation* @ q' M* T- |1 \& W y+ L
4. Unit 7.3 Metabolic Labeling with Sulfate
+ M% ]% [. B: m5 U+ `9 ]5. Unit 7.4 Metabolic Labeling with Fatty Acids
# ^/ s3 K8 H3 h; T/ W+ A6. Unit 7.5 Metabolic Labeling of Prenyl and Carboxyl-Methyl Groups i; O# g' u! y$ ]
7. Unit 7.6 Metabolic Labeling and Immunoprecipitation of Yeast Proteins$ R) Q2 Y6 K& N# i) b# ?
8. Unit 7.7 Metabolic Labeling and Immunoprecipitation of Drosophila Proteins
3 U; R. C8 `0 X. a4 l9. Unit 7.8 Metabolic Labeling of Glycoproteins with Radioactive Sugars
3 I; z M( J% I4 K- s4 M$ [, m10. Unit 7.9 Analysis of Oxidative Modification of Proteins3 K4 H3 n2 U1 W; o
11. Unit 7.10 Radioiodination of Cellular Proteins( T% [; g7 u: Y" J" c
10. Chapter 8 Cell Cycle Analysis
" i0 H$ R2 y, r* H) J& g1. Introduction
0 i- K0 i% k- ?$ ?6 _3 M* b$ n9 O2. Unit 8.1 Overview of the Cell Cycle
: s# r/ {6 _0 q3 Q, d3. Unit 8.2 Assays for CDK Activity and DNA Replication in the Cell Cycle
3 J% e0 i `" T7 u3 T4. Unit 8.3 Methods for Synchronizing Cells at Specific Stages of the Cell Cycle7 y8 x; w1 c4 }. s, P- E& n
5. Unit 8.4 Determining Cell Cycle Stages by Flow Cytometry! `8 o5 _( x; _/ V8 S, ^) ~
6. Unit 8.5 Centrifugal Elutriation to Obtain Synchronous Populations of Cells6 B/ \. A3 |$ @" b: ^! e7 P/ ~9 f
7. Unit 8.6 Dynamic Proliferation Assessment in Flow Cytometry8 ?% i) N) d( D, T/ I
11. Chapter 9 Cell Adhesion q8 L4 ] M' V; o
1. Introduction2 C* z! {; u9 `. m( `( Q) U, p. Z
2. Unit 9.1 Cell-Substrate Adhesion Assays
0 h8 w' i1 Y6 {/ s! W3. Unit 9.2 Quantitative Measurement of Cell Adhesion Using Centrifugal Force
8 m. ]9 F$ k1 b( I+ |5 T" G8 H- ~. n4. Unit 9.3 Cadherin-Dependent Cell-Cell Adhesion
" L4 `6 e0 a9 y0 x* `5. Unit 9.4 Analyzing Integrin-Dependent Adhesion
' s0 Z2 j$ O3 v* m( e+ d3 ~3 G6. Unit 9.5 Analysis of Cell-Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules
8 E% {. ], X7 ?( I, M' G5 B/ J2 K7. Unit 9.6 Measurement of Adhesion Under Flow Conditions
/ X! y) @: ?4 a8 f( V/ {2 W' G12. Chapter 10 Extracellular Matrix
+ g7 }8 i, b. e8 ?: ]! p1. Introduction
: O5 M3 R3 i& Y1 E0 [3 h/ W2. Unit 10.1 Overview of Extracellular Matrix5 T1 Z! ]( m/ `" _& X: w2 R
3. Unit 10.2 Preparation of Basement Membrane Components from EHS Tumors& Y" p' a% _( C# [& }+ _7 v$ i( L
4. Unit 10.3 Preparation of Gelled Substrates
: O$ h- A+ z M' X# m0 q5. Unit 10.4 Preparation of Extracellular Matrices Produced by Cultured Corneal Endothelial and PF-HR9" J, L0 j$ o6 o8 u# N# \# O
Endodermal Cells
9 p4 `; z) W# L3 r m: ^6. Unit 10.5 Purification of Fibronectin
$ Y6 H/ c1 b; k" X: h5 V# n# ~7. Unit 10.6 Purification of Vitronectin) A6 }) q" `8 k9 x# X; a) j3 b5 U( E
8. Unit 10.7 Proteoglycan Isolation and Analysis( o7 \" n; F: o
9. Unit 10.8 Matrix Metalloproteinases$ r7 K. [/ ?+ z- P# R* ~
10. Unit 10.9 Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts3 Z) q# K' O* O2 c8 Y5 T( |
11. Unit 10.10 Purification and Analysis of Thrombospondin-1
( Y1 X5 s. O: @5 P& y" F) ^12. Unit 10.11 Purification of SPARC/Osteonectin
% O5 q f4 g% H) I" D! N* K. _13. Unit 10.12 Analysis of Fibronectin Matrix Assembly
5 {5 q% K9 d, W ]; i1 ^+ P% M14. Unit 10.13 Non-Radioactive Quantification of Fibronectin Matrix Assembly0 y- V$ B/ Z. Y& a
15. Unit 10.14 Use of Hyaluronan-Derived Hydrogels for Three-Dimensional Cell Culture and Tumor! {7 Z( R, o' T1 Q3 d
Xenografts9 j5 [8 \ G# O5 j5 J O9 `
16. Unit 10.15 Generation of Micropatterned Substrates Using Micro Photopatterning9 S X' } M* m
17. Unit 10.16 Preparation of Hydrogel Substrates with Tunable Mechanical Properties3 W5 P1 q6 H/ L! V+ ]
18. Unit 10.17 Engineering Three-Dimensional Collagen Matrices to Provide Contact Guidance during 3D5 t ]9 M/ {8 l
Cell Migration! k7 ^7 Z; ]% w2 T* u! j+ R
19. Unit 10.18 Imaging Cells in Three-Dimensional Collagen Matrix5 A9 p2 ~+ s, }7 u1 l, v- F" |
13. Chapter 11 In Vitro Reconstitution
! a6 g; Y3 ^7 Y( S4 g- \' y1. Introduction: q$ \2 ~. {9 F0 D
2. Unit 11.1 Overview of Eukaryotic In Vitro Translation and Expression Systems$ R4 g, z9 I. j) d+ X1 `' O
3. Unit 11.2 In Vitro Translation
0 y' v, P$ V0 c0 C4. Unit 11.3 In Vitro Analysis of Endoplasmic-Reticulum-to-Golgi Transport in Mammalian Cells) Y9 Q2 a/ K. C. X1 K0 g* h
5. Unit 11.4 Cotranslational Translocation of Proteins into Canine Rough Microsomes! d0 Z8 _3 F" G! w8 F" B
6. Unit 11.5 In Vitro Analysis of SV40 DNA Replication" u ` @$ P- ~
7. Unit 11.6 In Vitro Transcription
* Z/ Q: Q# q) u9 B+ `8. Unit 11.7 Nuclear Import in Digitonin-Permeabilized Cells
) s- P6 a4 a) |$ ]7 A8 K9. Unit 11.8 In Vitro Translation Using HeLa Extract* I; r' D! U' B5 D
10. Unit 11.9 Analysis of Eukaryotic Translation in Purified and Semipurified Systems
|" y' C* c; q# h, \. r8 ^6 N11. Unit 11.10 Preparation and Use of Interphase Xenopus Egg Extracts
( A8 I$ E) t- r! o( [, Q: k& L0 |8 H12. Unit 11.11 Analysis of the Cell Cycle Using Xenopus Egg Extracts% x. P- q# i$ _9 `
13. Unit 11.12 Analysis of Apoptosis Using Xenopus Egg Extracts
# I9 G8 M( L: i4 ?; Q, n14. Unit 11.13 Mitotic Spindle Assembly In Vitro
( H/ ]" _4 Z- m15. Unit 11.14 Analysis of RNA Export Using Xenopus Oocytes2 {* E% ^$ r/ j/ x- X
16. Unit 11.15 In Vitro Analysis of Peroxisomal Protein Import
& G& [3 f- D, Z ^5 ~& `" y% U17. Unit 11.16 In Vitro Analysis of Chloroplast Protein Import
3 Q6 b- C8 Z4 G9 m* p R- F18. Unit 11.17 In Vitro RNA Splicing in Mammalian Cell Extracts
% `0 |' t; f2 n* u0 }+ }19. Unit 11.18 Endocytosis Assays in Intact and Permeabilized Cells2 a( A: o7 j2 t6 w( m' o# W& k1 g
20. Unit 11.19 In Vitro Analysis of Yeast Mitochondrial Protein Import, {8 H8 ~( G2 o5 Z+ T
14. Chapter 12 Cell Motility
0 |5 S9 n+ T8 Y5 a& y& M/ r9 z1. Introduction
, p) q& y" M- b `' u J! P, g2. Unit 12.1 Chemotaxis Assays for Eukaryotic Cells( u5 C0 { Z6 M" H
3. Unit 12.2 Invasion Assays7 g: ~' [4 z2 e1 e+ N w" r
4. Unit 12.3 Cell Traction l. y% L6 f- t) F/ E
5. Unit 12.4 Cell Wound Assays, h: t# F0 x% O3 q, Q0 _
6. Unit 12.5 Dictyostelium Cell Dynamics
% {, G1 O4 v( \4 A7. Unit 12.6 Optical Microscopy.Based Migration Assay for Human Neutrophils. R B' f6 s0 W1 v1 @
8. Unit 12.7 Actin-Based Motility Assay
2 D$ s$ I' e; Y8 r C" R* S9. Unit 12.8 In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
3 o1 j) ^% s* U& z D. r; ] A4 E15. Chapter 13 Organelle Motility, V, i7 z9 {$ }' l* q
1. Introduction# s) M. j0 a7 o9 m) K _
2. Unit 13.1 Microtubule/Organelle Motility Assays
/ l* m; R4 \, q3 C: B3. Unit 13.2 In Vitro Motility Assay to Study Translocation of Actin by Myosin
, L% ~: u8 O# O; {1 W, [ n* l4. Unit 13.3 Organelle Motility in Plant Cells: Imaging Golgi and ER Dynamics with GFP5 d! M0 t6 U6 p/ V
5. Unit 13.4 Movement of Nuclei3 x* l8 z7 v |8 I# g
6. Unit 13.5 Measuring Dynamics of Nuclear Proteins by Photobleaching( y! w B) t* H: B; n
7. Unit 13.6 Functional Characterization of Proteins Regulating Actin Assembly# g) _# s* A$ q4 O" x- Y
16. Chapter 14 Signal Transduction: Protein Phosphorylation9 U: q! T: X. }+ C
1. Introduction
7 W$ _8 N' e7 w1 L8 \4 l2. Unit 14.1 Overview of Protein Phosphorylation
2 V' R- P; o/ k+ K. j! ~3. Unit 14.2 Immunological Detection of Phosphorylation5 H9 d: K- |/ E' Y N! N, Z
4. Unit 14.3 The Detection of MAPK Signaling
5 P2 I2 j9 a, _! x, R5. Unit 14.4 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
0 {" U5 a6 p1 x' [, R9 U: v6. Unit 14.5 Phosphoamino Acid Analysis, s1 K$ ~9 k+ E" X$ x9 e! z0 L
7. Unit 14.6 Determination of Akt/PKB Signaling
7 \3 M5 i# O/ r( t$ M# ^% v+ K9 j4 c8. Unit 14.7 Analyzing FAK and Pyk2 in Early Integrin Signaling Events: N/ C% [: E4 T5 D9 @
9. Unit 14.8 Rho GTPase Activation Assays
i% t" V8 R0 B0 R$ G9 f) `) P4 B10. Unit 14.9 In Vitro GEF and GAP Assays g& k) ] q2 p) C- o: J% y# k
11. Unit 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Forster Resonance
* e3 i( \' v0 z; J7 AEnergy Transfer (FRET)
8 b* T0 A, s# { w12. Unit 14.11 Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells9 W# m; `2 B3 F# @, t0 `% S! f! t
13. Unit 14.12 Analysis of Arf GTP-Binding Protein Function in Cells* Z- W- o4 X7 J) z5 b8 L! U
17. Chapter 15 Protein Trafficking
9 A+ ~ u" Z* f7 N+ Q1 s1. Introduction8 ^( b. Z! w$ T1 I2 |
2. Unit 15.1 Overview of Protein Trafficking in the Secretory and Endocytic Pathways
0 B4 X" a, F2 A( N4 U- c- O3. Unit 15.2 Use of Glycosidases to Study Protein Trafficking: E5 P1 f0 v# M8 g6 q- r
4. Unit 15.3 Endocytosis: Biochemical Analyses1 q1 y; u6 D; t3 {
5. Unit 15.4 Determining Protein Transport to the Plasma Membrane# I. J) y/ W' h6 e
6. Unit 15.5 Analysis of Membrane Traffic in Polarized Epithelial Cells
& ^( O4 p- q8 ]+ f7. Unit 15.6 Analysis of Protein Folding and Oxidation in the Endoplasmic Reticulum
7 t/ K( d8 z p1 S( A5 J( j7 Z8. Unit 15.7 Measurements of Phagocytosis and Phagosomal Maturation+ ]+ Q8 L% G; G2 M: h" V2 s) y9 A
9. Unit 15.8 Analysis of Protein Transport to Lysosomes3 H9 \+ H4 I7 ?- n U8 P& ?
10. Unit 15.9 Studies of the Ubiquitin Proteasome System: T3 S3 f5 B( t
11. Unit 15.10 Measuring Retrograde Transport to the Trans-Golgi Network0 N( g0 O' V$ V9 v5 r+ V& Y
12. Unit 15.11 Assays for Regulated Exocytosis of Mast Cell Granules! [' P% |* B5 |* I8 \
13. Unit 15.12 Analysis of Regulated Secretion Using PC12 Cells
W' k/ I. L% H' E, b, s14. Unit 15.13 Analysis of Endocytic Trafficking by Single-Cell Fluorescence Ratio Imaging! g6 i. Q+ T- Y# Y' R
15. Unit 15.14 Quantitative Analysis of Endocytosis and Turnover of Epidermal Growth Factor (EGF) and
/ R# m/ l! X, y( P& Z- sEGF Receptor. X& `5 q( P* w' t1 p7 b( m
16. Unit 15.15 Documenting GLUT4 Exocytosis and Endocytosis in Muscle Cell Monolayers6 d& c( v( t& P+ v
18. Chapter 16 Antibodies as Cell Biological Tools% K# V' F: L. X7 S! C4 \* y
1. Introduction
5 H. z/ _9 L' B. F! z2. Unit 16.1 Production of Monoclonal Antibodies
7 u0 [9 `# r1 P5 t, o% ]/ L2 ~* Q3. Unit 16.2 Production of Polyclonal Antisera
/ ]: ?& m4 Q" k k' V4. Unit 16.3 Purification of Immunoglobulin G& ]7 Y6 \/ h4 Z
5. Unit 16.4 Fragmentation of Immunoglobulin G
& z7 D2 b& B( s6. Unit 16.5 Antibody Conjugates for Cell Biology
$ e" T& f" d" i0 D7. Unit 16.6 Production of Antibodies That Recognize Specific Tyrosine-Phosphorylated Peptides" B, {9 n8 ?+ U$ J
19. Chapter 17 Macromolecular Interactions in Cells
: D$ s* ?; k, M, E/ e$ D* {+ I) R1. Introduction f T' Q1 O# u$ S6 Y1 ? S
2. Unit 17.1 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
3 G$ l, |4 a' h x6 DMicroscopy6 Z& A4 t) {6 c6 U9 {9 L* T
3. Unit 17.2 Identification of Protein Interactions by Far Western Analysis
( C/ x" |0 g7 T, K- C4. Unit 17.3 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
4 \; J4 Y$ I- y0 j' z) T r3 j5. Unit 17.4 Mapping Protein-Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries
, |9 g/ u, \8 Q& ^6. Unit 17.5 Protein-Protein Interactions Identified by Pull-Down Experiments and Mass Spectrometry+ W" k7 { d6 i5 B" r7 I
7. Unit 17.6 Measuring Protein Interactions by Optical Biosensors: O4 E, P& s) H! C; O
8. Unit 17.7 Chromatin Immunoprecipitation for Determining the Association of Proteins with Specific
; Z9 V8 Z6 N3 c* u/ \$ cGenomic Sequences In Vivo) f5 z+ R( z! i
9. Unit 17.8 Isothermal Titration Calorimetry( O! j6 Y- F* V! ~% a; o k
10. Unit 17.9 Rational Design and Evaluation of FRET Experiments to Measure Protein Proximities in Cells/ T0 j8 E& J( F, v
11. Unit 17.10 Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking) t" D5 s1 Q- ? T6 x. t
12. Unit 17.11 Visualization of RNA Using Fluorescence Complementation Triggered by Aptamer-Protein
/ I0 H: v% D3 t& m# gInteractions (RFAP) in Live Bacterial Cells
1 e8 A" n# L2 t2 I$ S2 i. c20. Chapter 18 Cellular Aging and Death& W0 W7 S( Z4 r, r i3 |9 b% S, W
1. Introduction6 ?/ Q& i8 E# W! m4 E9 J" O( k y
2. Unit 18.1 Current Concepts in Cell Death9 p4 v& y) N4 r, @- e
3. Unit 18.2 Analysis of Caspase Activation During Apoptosis
4 `; P) G" @/ k" K L( e+ \. n6 f# d4. Unit 18.3 Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria5 m9 K% X3 c& V% J- u- A
5. Unit 18.4 Quantitative Fluorescence In Situ Hybridization (Q-FISH)
" ~+ e5 o& l' T2 k6. Unit 18.5 Analysis of Mitochondrial Dysfunction During Cell Death. t4 `2 s7 D2 y' o n
7. Unit 18.6 Analysis of Telomeres and Telomerase
6 E3 d9 F) C o4 s' [0 d& H# a8. Unit 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of
2 h2 c0 c: O5 H& G' ?+ kPoly(ADP-Ribose) Polymerase: Z0 C, _2 K0 A- Y+ ^9 b
9. Unit 18.8 Flow Cytometry of Apoptosis
% x4 ^8 u8 u3 V6 W10. Unit 18.9 Analysis of Cellular Senescence in Culture In Vivo: The Senescence-Associated -Galactosidase1 G" ?& p" d! x
Assay
- G$ e5 \9 o. q, A0 X11. Unit 18.10 High-Throughput Live Cell Imaging of Apoptosis$ h! [4 a9 H- ]
21. Chapter 19 Whole Organism and Tissue Analysis4 {1 T( S1 O/ _" S" F. j8 P
1. Introduction Z; m: Y( Z" R q; ]" C+ m2 l
2. Unit 19.1 Overview of Metastasis Assays
! ?7 ?/ x& @7 Z, q3. Unit 19.2 Tail Vein Assay of Cancer Metastasis
( [& t {! I1 z/ u0 o4. Unit 19.3 Microanalysis of Gene Expression in Tissues Using T7-SAGE: Serial Analysis of Gene X# }2 G: @ h* \
Expression After High-Fidelity T7-Based RNA Amplification
D+ X" L) C# m4 N0 `% M% W9 k5. Unit 19.4 SAGE Analysis from 1 兪g of Total RNA; x7 H# i8 J8 \' k# G
6. Unit 19.5 The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
; w1 B Z+ x' w8 |# Q) H7. Unit 19.6 Experimental Metastasis Assays in the Chick Embryo; d0 [3 N! T9 U- J' o
8. Unit 19.7 Imaging Tumor Cell Movement In Vivo
- s; C% ~5 I+ ]( h) E* d% M* ]9. Unit 19.8 Embryonic Organ Culture
, P, d4 }% U) x* U10. Unit 19.9 Three-Dimensional Tissue Models of Normal and Diseased Skin
" r- W- u* C1 b11. Unit 19.10 Overview: Engineering Transgenic Constructs and Mice) A7 h+ D. P, [* _5 J$ r
12. Unit 19.11 Generation of Transgenic Mice
) {8 ~/ L5 Z0 ~3 t7 m! N' |13. Unit 19.12 Overview: Generation of Gene Knockout Mice% M# ? D0 \8 o6 ~& @1 J6 x9 S
14. Unit 19.13 Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production
7 b6 v! T! b( j2 V15. Unit 19.14 Generation of Gene Knockout Mice by ES Cell Microinjection/ _" c2 ]; Q: X+ J
22. Chapter 20 Expression and Introduction of Macromolecules into Cells
% v5 x7 _7 B$ C D1. Introduction! w& m- O; n5 a8 Z1 ~1 ^1 \
2. Unit 20.1 Direct Introduction of Molecules into Cells* x# M' I0 ]3 l5 {* }0 G" e3 G3 L
3. Unit 20.2 Protein Transduction: Generation of Full-Length Transducible Proteins Using the TAT System
0 v9 R, J: v: q4. Unit 20.3 Calcium Phosphate Transfection4 c" [& G8 N; t' @% C. C
5. Unit 20.4 Transfection Using DEAE-Dextran7 D& Z$ @8 O+ A* I# S
6. Unit 20.5 Transfection by Electroporation
7 s5 s, y* z C1 X7. Unit 20.6 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents$ E1 `" m$ @6 h. O
8. Unit 20.7 Optimization of Transfection
; J4 y: B+ \. d& M, E* P9. Unit 20.8 Inducible Gene Expression Using an Autoregulatory, Tetracycline-Controlled System! a7 z M# G$ A' @$ r0 _
23. Chapter 21 Fluorescent Protein Technology
9 r- q/ t( h2 F2 o$ _: [) A. }1. Introduction/ {# f2 c- K) {+ j' b! }. h# Y
2. Unit 21.1 Measuring Protein Mobility by Photobleaching GFP Chimeras in Living Cells
) \5 R) k/ i& U) K$ K* s3. Unit 21.2 Fluorescence Localization After Photobleaching (FLAP)8 }! d' |9 w' V7 `
4. Unit 21.3 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
& I2 Q; c/ r. y8 l5 p+ @Complementation (BiFC) Analysis8 h" b3 M9 Y, A+ k0 E6 B
5. Unit 21.4 Design and Use of Fluorescent Fusion Proteins in Cell Biology5 C, q g( p. A$ r) r# ^5 J3 v
6. Unit 21.5 The Fluorescent Protein Color Palette
) `7 Z' b) C% X0 H4 L7. Unit 21.6 Photoactivation and Imaging of Photoactivatable Fluorescent Proteins
9 T& Y0 M) ` i) }6 F# a24. Chapter 22 Cell Biology of Chromosomes and Nuclei8 m2 y! e! w" w/ d5 }3 x* s
1. Introduction! K0 ^8 F$ q A# I1 p
2. Unit 22.1 Overview of Cytogenetic Chromosome Analysis
8 l) G/ j( ]* T' U# g3. Unit 22.2 Preparation of Cytogenetic Specimens from Tissue Samples
# I- w) c9 f- s3 j8 H* k- ]' j4. Unit 22.3 Traditional Banding of Chromosomes for Cytogenetic Analysis# V8 H, _! t u) k( [
5. Unit 22.4 Fluorescence In Situ Hybridization (FISH)) O: p) x( l7 Q! a% E
6. Unit 22.5 Multi-Color FISH Techniques
5 e7 _* R; E1 y7. Unit 22.6 Comparative Genomic Hybridization2 z" u1 ~$ L0 I7 l6 x
8. Unit 22.7 Sister Chromatid Exchange
7 t) g4 p" @; O9 Z1 h1 \9. Unit 22.8 Detection of Mitotic Figures and Components of the Mitotic Machinery
) u% W9 V4 O% n7 {9 J10. Unit 22.9 Assembly and Micromanipulation of Xenopus In Vitro.Assembled Mitotic Chromosomes
s# P7 f% z& Z- K {1 L6 G& k11. Unit 22.10 Replication Labeling with Halogenated Thymidine Analogs+ ~2 q+ O2 n) ?( V
12. Unit 22.11 Assays for Ribosomal RNA Processing and Ribosome Assembly6 T# ? l/ i2 ?
13. Unit 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells* G/ z4 x; o4 ]$ D" {7 n
14. Unit 22.13 Monitoring mRNA Export
# R. h% h/ T) _! E: S) K3 i' R/ C15. Unit 22.14 Analysis of DNA Replication in Saccharomyces cerevisiae by Two-Dimensional and Pulsed-, ^6 y5 C) j% [* m0 x! z1 ^, Q
Field Gel Electrophoresis
! N5 `2 |/ {* c- P25. Chapter 23 Stem Cells2 D7 D" E# S/ i: P
1. Introduction
9 V& r$ m, L6 T2. Unit 23.1 Stem Cells: An Overview: t; I2 Z y! P- Y- N6 _
3. Unit 23.2 Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture( c+ d8 m% d0 X+ t6 M" A) p, ]) J2 a
and Differentiation as Embryoid Bodies
2 p" C7 `; I& \7 q& p4. Unit 23.3 Maintenance and In Vitro Differentiation of Mouse Embryonic Stem Cells to Form Blood
5 J, c% Q3 D: c( @Vessels
i9 Y' A" ?. k1 G2 P5. Unit 23.4 Differentiation of Mouse Embryonic Stem Cells and of Human Adult Stem Cells into2 I4 Z/ E; n+ K% S7 J7 E
Adipocytes- L, j3 a& b V U
6. Unit 23.5 Induction of ES Cell.Derived Cartilage Formation5 h, P6 F" e7 f: S2 M! o
7. Unit 23.6 Hematoendothelial Differentiation of Human Embryonic Stem Cells. E2 F5 d' v; @' [2 y0 q
8. Unit 23.7 Neural Differentiation of Human ES Cells" m$ g/ y: e0 L# h
26. Chapter 24 Lipids
; I& C1 V; L3 f$ J- I: p1. Introduction7 q1 U1 _7 @# i- d
2. Unit 24.1 Using Fluorescent Sphingolipid Analogs to Study Intracellular Lipid Trafficking
6 E- S6 n% _2 S Y- H2 ]0 F$ s3. Unit 24.2 Fluorescent Detection of Lipid Droplets and Associated Proteins0 P3 c% I0 V- h+ s
4. Unit 24.3 Making Giant Unilamellar Vesicles via Hydration of a Lipid Film: F {, y) B/ k4 v* i( ^0 W3 {
5. Unit 24.4 Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains: i& M4 m% i5 v8 s5 Y0 t- G- z4 t2 v0 a
27. Chapter 25 Nanotechnology
' O3 W# B. |6 r# f" R/ B; ]; k# s1. Introduction
% c7 g2 V% V! ^- \, X- g! M, `2. Unit 25.1 In Vivo Imaging Using Quantum Dot.Conjugated Probes3 d- W: C. l g) i: a6 [
3. Unit 25.2 Fabrication and Application of Nanofibrous Scaffolds in Tissue Engineering5 _) Y% T0 B" a6 y( q1 ]
28. Chapter 26 Viruses1 E) g- \$ p- e& w$ x- N
1. Introduction
- Y9 o# S8 m5 {* j2. Unit 26.1 Production of Papillomavirus-Based Gene Transfer Vectors$ y( i; Q- x5 s8 |5 u4 O# ?
3. Unit 26.2 BK Virus (BKV): Infection, Propagation, Quantitation, Purification, Labeling, and Analysis of
9 I5 A% P, \9 F% T* d DCell Entry
- H% C7 z/ Q. p" }0 G- D- p4. Unit 26.3 Methods Used to Study Respiratory Virus Infection% r5 c, c) ~2 a
5. Unit 26.4 Compartmented Neuron Cultures for Directional Infection by Alpha Herpesviruses& i+ M) Z" p" D4 V1 i
6. Unit 26.5 HIV-1 Interactions with Cells: From Viral Binding to Cell-Cell Transmission6 b/ V Z% M2 ^# f" J. q0 ^
29. Chapter 26 Lipids
0 H. Z3 Y" e7 A2 c" {1. Unit 26.6 Methods for Monitoring Dynamics of Pulmonary RSV Replication by Viral Culture and by
+ Y* u: ~# H) \2 H3 m1 p' Q# P) rReal-Time Reverse Transcription.PCR In Vivo: Detection of Abortive Viral Replication
" a- r2 S4 j/ a% R) R30. Chapter 27 RNA-Based Methods in Cell Biology8 d' x! m2 d% k r; R3 c' |; ^
1. Introduction
6 x! q' N {" I$ |) Y1 _2. Unit 27.1 Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs
$ ^0 {3 \. M |. @# {3. Unit 27.2 Gene Down-Regulation with Short Hairpin RNAs and Validation of Specificity by Inducible
2 s3 Q+ t3 L1 z3 Z( K0 RRescue in Mammalian Cells
! l1 R% x/ [" ?. m31. Appendix 1 Useful Information and Data
1 @5 t2 k. d) N1. 1A Useful Measurements and Data! w: p! \. u" X O
2. 1B Compendium of Drugs Commonly Used in Cell Biology Research3 [( x2 ?) S+ \0 N' T
3. 1C Identification of Motifs in Protein Sequences. c+ E# k1 e. n0 z, K$ b: l
4. 1D Safe Use of Radioisotopes
- x' x# C5 S$ E3 j3 y/ A2 A9 ^( C1 Y5. 1E Absorption and Emission Maxima for Common Fluorophores1 Z! [) s% E5 V3 e8 q
6. 1F Importing Biological Materials2 }; b' L0 `. N/ V
7. 1G Centrifuges and Rotors
& D1 m J o2 B& e! _8. 1H Internet Basics for Biologists
- l6 a' }7 c: o/ |32. Appendix 2 Laboratory Stock Solutions and Equipment
: T$ k b0 q. p7 y& [4 r1. 2A Common Stock Solutions, Buffers, and Media
! d p' F* d x( m2. 2B Medium Formulations, L+ |4 X, R! S3 j }
3. 2C Standard Laboratory Equipment
) x2 g# U( O O: u. r8 L! \33. Appendix 3 Commonly Used Techniques
; X# T0 R s5 R1. 3A Molecular Biology Techniques
8 Q$ Q$ M' N% ~# P0 S. ^2. 3B Spectrophotometric Determination of Protein Concentration
0 m4 Q; t" C1 w3. 3C Dialysis and Concentration of Protein Solutions" k" y8 E, b: k
4. 3D Quantification of DNA and RNA with Absorption and Fluorescence Spectroscopy
6 \' K# a& Q0 D% r- t2 r5. 3E Silanizing Glassware/ l- w, H! ]% _% \8 _8 R
6. 3F Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
6 l5 s8 W( P9 X( F* E2 X( l7. 3G Micro RT-PCR2 _) j% y1 J6 M) @0 d! b0 G) ?) D6 j, q
8. 3H The Colorimetric Detection and Quantitation of Total Protein
! q2 S* J7 ?* K* r7 {1 s# [& b34. Appendix Suppliers: `/ b" k" C; S0 V' Q* v
1. Selected Suppliers of Reagents and Equipment4 u, `& i+ p- U6 w. v6 Z+ \, ]! E2 @
% [/ e8 Z. o0 C/ D6 e7 \
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发表于 2011-3-8 15:53
