* Contains more than 40% new material
* Provides cell biologists and other life scientists with the most up-to-date instructions for basic and advanced cell biological techniques, including those at the interface between cell and molecular biology
* Features uniform style and editing and includes contributions from world-renowned authorities in their respective fields
* Contains information appropriate for a large, diverse, and constantly growing international audience of cell, developmental, and molecular biologists, plus others who need these methods in their laboratory research
* Includes color plates throughout the set for easy reference
* Designed as the essential lab guide and research reference for the field
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From the Introduction
I. INTRODUCTION The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has generated immense interest because it offers a means to mark and visualize cells and proteins noninvasively. Thus GFP can be used in living cells and organisms. GFP has three attractive properties as a fluorescent marker: (1) absorption and emission wavelengths of the chromophore are in the visible region; (2) because the fluorescence is intrinsic to the molecule, the protein forms the chromophore in an autocatalytic reaction and does not require any cofactors; and (3) the cDNA for GFP has been cloned (Prasher, 1992), therefore it can be expressed in any cell type and used as a fluorescent "tag."
The GFP cDNA has been expressed in a variety of organisms, including bacteria, nematodes, yeast, Dictyostelium, Xenopus, Drosophila, plants, mice, and mammalian cell lines. GFP has been used as a marker to determine cell fates in development, as a reporter for gene expression, and as a tag to follow protein localization (for a review see Cubitt et al. 1995).
This article will mainly describe using GFP as a localization marker in living mammalian cells.
A. Structure of GFP GFP is a 27-kDa protein (238 amino acids). The chromophore in GFP is created by posttrans-lational modification: an autocatalytic cyclization and oxidation of the tripeptide Ser65-Tyr66-Gly67. The time taken before GFP-tagged proteins can be observed in cells is mainly determined by the rate of protein folding and cyclization. The mature protein must fold correctly to generate the chromophore, and correct folding appears to be temperature dependent. Once formed, however, the protein resists extensive protease treatment and temperatures up to 657C. The basis for this remarkable stability is revealed in the recently solved crystal structure, which shows that GFP forms a cylinder composed of b sheets, with the chromophore contained within the well-protected core of the protein (Yang et al., 1996; Ormö et al., 1996).
B. Spectral Properties Wild-type GFP has a major excitation peak at 395 nm, a minor peak at 475 nm, and emits green light with a peak at 509 nm. The fluorescent properties of wild-type GFP are sufficient for some applications, notably in those organisms that live at temperatures similar to A. victoria. Conventional fluorescein-optimized filter sets can be used to visualize wild-type GFP, which is then excited at the minor 475 nm peak. When excited at 395 nm, photoisomerization causes a gradual decrease in the 395 nm peak and a corresponding increase in absorption at 475 nm. However, the fluorescence emitted by wild-type GFP is weak and it bleaches relatively fast. Several mutant GFPs have now been generated with improved spectral charac-teristics. These GFP variants contain mutations that alter absorption or emission spectra, or improve protein folding and thus influence the fluorescence intensity.
Table of ContentsVolume Four:
Part 13: Transfer of Macromolecules and Small Molecules: Section A: Microinjection Using Glass Capillaries:
M.R. Jacobson, T. Pederson, and Y. Wang, Microinjection of RNA and DNA into Somatic Cells.
M. Graessmann and A. Graessmann, Microinjection of RNA and DNA into Somatic Cells.
R. Pepperkok, R. Saffrich, and W. Ansorge, Computer-Automated and Semiautomated Capillary Microinjection of Macromolecules into Living Cells.
Y. Fukui, Microinjection into Dictyostelium Amoebae.
G. Matthews, Microinjection of RNAs into Xenopus Oocytes.
Section B: Syringe Loading:
M.S.F. Clarke and P.L. McNeil, Syringe Loading: A Method for Inserting Macromolecules into Cells in Suspension.
Section C: Electroporation:
S. Herr, R. Pepperkok, R. Saffrich, S. Wiemann, and W. Ansorge, Electroporation of Cells.
H.L. Brownell and L. Raptis, Electroporation of Nucleotides: 32P Labeling of Cellular Proteins or Assessment of Ras Activity.
L. Raptis, H.L. Brownell, K.L. Firth, and S.G.-Peraldi, In Situ Electroporation for the Study of Signal Transduction.
M.G. Ormerod, Monitoring Electroporation by Flow Cytometry.
W. Dillen, M. Van Montagu, and G. Angenon, Electroporation-Mediated DNA Transfer to Plant Protoplasts and Intact Plant Tissues for Transient Gene Expression Assays.
Section D: Pore-Forming Toxins and Other Procedures:
G. Ahnert-Hilger and U. Weller, a-Toxin and Streptolysin O as Tools in Cell Biological Research.
A. Joliot, D. Derossi, S. Calvet, and A. Prochiantz, Homeodomain and Homeodomain-Derived Peptide: New Vectors for Internalization of Molecules into Living Cells.
Section E: Liposomes and Lipofection:
Y. Saeki and Y. Kaneda, Protein-Modified Liposomes (HVJ-Liposomes) for the Delivery of Genes, Oligonucleotides, and Proteins.
G. Gregoriadis, B. McCormack, Y. Morrison, R. Saffie, and B. Zadi, Liposomes in Drug Targeting.
H. Stenmark and M. Zerial, Lipofection.
M.L. Tilkins, P. Hawley-Nelson, and V. Ciccarone, Transfection of Mammalian and Invertebrate Cells Using Cationic Lipids.
Section F: Microprojectile Bombardment:
J.R. Kikkert, Biolistic Transformation of Plant Cells.
G. Spangenberg and Z. Wang, Biolistic Transformation of Embryogenic Cell Suspensions.
Section G: Transformation of Plant Cells:
R. Bilang and I. Potrykus, Protoplast Uptake of DNA.
S.H. Park, D. Kirubi, C. Zapata, M. Srivatanakul, T.S. Ko, S. Bhaskaran, S. Rose, and R.H. Smith, Monocot and Dicot Transformation Using Agrobacterium tumefaciens and the Shoot Apex.
Part 14: Expression Systems: Section A: Cell-Free Systems:
P. Madsen, P. Gromov, and J.E. Celis, Expression of cDNA Clones by Coupled In Vitro Transcription/Translation from Plasmids Containing Viral Transcription Promoters.
G. Matthews, Preparation and Use of Translocating Cell-Free Translation Extracts from Xenopus Eggs.
Section B: Eukaryotic Expression Systems:
H. Stenmark and M. Zerial, Transient Expression Using the T7 RNA Polymerase Recombinant Vaccinia Virus System.
L.A. King, S.A. Marlow, L.E. Wilson, and R.D. Possee, The Baculovirus Expression Vector System I: Production and Isolation of Recombinant Viruses.
L.A. King, S.A. Marlow, L.A. Obosi, and R.D. Possee, Baculovirus Expression Vector System II: Amplification and Characterization of Recombinant Viruses.
M. Ekström, H. Garoff, and H. Andersson, Semliki Forest Virus Expression System.
P. Gromov, J.E. Celis, and P. Madsen, Transient Expression of cDNAs in COS-1 Cells and Protein Analysis by Two-Dimensional Gel Electrophoresis.
S. Freundlieb, U. Baron, and H. Bujard, Controlling Gene Activities via Tetracycline Regulatory Systems.
J.D. Chestnut and J.P. Hoeffler, Rapid Selection of Transiently Transfected Cells from Culture Using the Capture-Tec System.
C. Karlsson and J. Pines, Green Fluorescent Protein.
Section C: Expressions of cDNAs in E. Coli:
M. Hyvönen and M. Saraste, Expression of cDNAs in Escherichia coli Using the T7 Promoter.
Section D: Expression System for Cytoskeletal Studies:
M. Gimona, Liposome-Mediated Transfection of REF-52 Cells.
Part 15: Differential Gene Expression:
M.H.K. Linskens, L.A. Tonkin, and S.M. Saati, Enhanced Differential Display: A Reproducible Method for the Analysis of Differential Gene Expression.
I.N. Hampson and T.M. Dexter, Chemical Crosslinking Subtraction.
Part 16: Proteins:
Section A: Protein Determination and Amino Acid Analysis:
M. Guttenberger, Protein Determination.
F. Lottspeich, C. Eckerskorn, and R. Grimm, Amino Acid Analysis on Microscale.
P. van der Geer, K. Luo, B.M. Sefton, and T. Hunter, Phosphopeptide Mapping and Phosphoamino Acid Analysis on Cellulose Thin-Layer Plates.
Section B: Preparation of Tagged Proteins:
M.J. Rudick, Controlled Radioiodination of Proteins.
A.D. Marmorstein, C. Zurzolo, A. Le Bivic, and E. Rodriguez-Boulan, Cell Surface Biotinylation Techniques and Determination of Protein Polarity.
B. Walker and A. McGinty, Detection and Labeling of Proteases Using Biotinylated Active Site-Directed Affinity Labels.
Section C: Gel Electrophoresis:
J.E. Celis and E. Olsen, One-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis.
D. Safer, Nondenaturing Polyacrylamide Gel Electrophoresis as a Method for Studying Protein Interactions.
J.E. Celis, G. Ratz, B. Basse, J.B. Lauridsen, A. Celis, N.A. Jensen, and P. Gromov, High-Resolution Two-Dimensional Gel Electrophoresis of Proteins: Isoelectric Focusing (IEF) and Nonequilibrium pH Gradient Electrophoresis (NEPHGE). Applications to the Analysis of Cultured Cells and Mouse Knockouts.
A. Görg and W. Weiss, High-Resolution Two-Dimensional Electrophoresis of Proteins Using Immobilized pH Gradients.
M. Gimona, B. Galazkiewicz, and M. Niederreiter, Mini Two-Dimensional Gel Electrophoresis.
J.E. Celis, G. Ratz, and B. Basse, Electroelution of Proteins from Two-Dimensional Gels
Section D: Labeling of Cells:
P. Gromov and J.E. Celis, Two-Dimensional Analysis of Posttranslationally Modified Proteins.
Section E: Gel Staining:
C.R. Merril, G.J Creed, and R.C. Allen, Ultrasensitive Silver-Based Stains for Protein Detection.
A. Wallace and H.P. Saluz, Enhanced Detection of Silver-Stained Proteins in Polyacrylamide Gels by Radioactivation.
Section F: Overlay Techniques and Others:
K.L. Schmeichel, M.C. Beckerle, and A.W. Crawford, Blot Overlay Assay: A Method to Detect Protein-Protein Interactions.
H.J. Hoffmann, P. Gromov, and J.E. Celis, Calcium Overlay Assay.
P.S. Gromov and J.E. Celis, Blot Overlay Assay for the Identification of GTP-Binding Proteins.
S. Bar-Nun and J.M. Gershoni, Protein-Blot Analysis of Glycoproteins and Lectin Overlays.
D.A. Shackelford, Detection of Protein Kinase Activity after Renaturation of Proteins in Sodium Dodecyl Sulfate-Polyacrylamide Gels or on Membranes.
C. Paech and T. Christianson, Zymography of Proteases.
K. Dejgaard and J.E. Celis, Two-Dimensional Northwestern Blotting.
Section G: Techniques to Reveal Interacting Proteins:
J. Lukas and J. Bartek, Immunoprecipitation of Proteins under Nondenaturing Conditions.
R. Graf, J. Brunner, B. Dobberstein, and B. Martoglio, Probing the Molecular Environment of Proteins by Site-Specific Photocrosslinking.
Section H: Peptide Microsequencing:
J. Vandekerckhove and H.H. Rasmussen, Internal Amino Acid Sequencing of Proteins Recovered from One- or Two-Dimensional Gels.
H. Nika and R. Aebersold, Amino-Terminal Protein Sequence Analysis.
Section I: Mass Spectrometry:
J.R. Yates, III, Peptide Sequencing by Tandem Mass Spectrometry.
J.R. Yates, III, E. Carmack, and J.K. Eng, Direct Database Searching Using Tandem Mass Spectra of Peptides.
B.L. Gillece-Castro, Mass Spectrometry: Detection and Characterization of Posttranslational Modifications.
P. Roepstorff, M.R. Larsen, H.R. Nielsen, and E. Nordhoff, Sample Preparation Methods for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Peptides, Proteins, and Nucleic Acids.
R.W. Davies, Working Safely with Radioactivity.
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