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`Handbook (cid:9) (cid:149)
`i and IResearch Products
`Ninth Edition by Richard P. Haugland, Ph.D.
`Jay Gregory, Ph.D., Editor
`Michelle TZ. Spence, Ph.D., Continuity Editor
`lain Johnson, Ph.D., Technical Information Coordinator
`Erik Miller, Content Management Software Developer
`
`2
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`
`
`Molecular Probes, Inc.
`29851 Willow Creek Rd.
`Eugene, OR 97402 USA
`Phone: (541) 465-8300
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`Web: www.probes.com
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`
`Editor
`Jay Gregory
`Continuity Editor
`Michelle T.Z. Spence
`Content Management Software Developer
`Erik Miller
`Editorial Assistance
`Aaron Basey
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`Rakar West
`Production Support
`Lynda J. Gansel (Production Supervisor),
`Cinch Brown, Stephanie Perkins,
`Kathleen Simpson, Miriam Sisson
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`Technical Information Coordinator
`lain Johnson
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`Editorial Contributors
`Kathleen E. Free (Chapters 1-5, 10, 16, 17, 19)
`Jill Hendrickson (Chapters 8, 9, 24)
`lain Johnson (Chapters 6, 13. 14, 18, 20-24)
`Chip Walker (Chapters 7, 11, 12, 15, 18)
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`Molecular Probes research and development staff.
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`This Handbook would not have been possible without the help of the dedicated staff at Molecular Probes,
`including those in the following departments: Accounting, Analytical Services, Biosciences, Business
`Development, Custom and Bulk Sales, Customer Service, Facilities, Human Resources, Information
`Systems, Legal, Marketing, Organic Chemistry, Packaging, Product Administration, Purchasing, Quality
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`Molecular Probes, Inc., Eugene, OR 97402; Founded in 1975.
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`Molecular Probes Europe By, Leiden, The Netherlands; Founded in 1995.
`
`' 2002 by Molecular Probes, Inc.
`
`First Edition published 1978. Second Edition 1981. Third Edition 1985. Fourth Edition 1989. Fifth
`Edition 1992. Sixth Edition 1996. Seventh Edition 1999 (on CD only). Eighth Edition 2000 (on CD only).
`Ninth Edition 2002.
`
`All rights reserved. No part of this book may be reproduced or used for commercial purposes in any form
`or by any means graphic, electronic or mechanical, including photocopying, recording, taping or informa-
`tion storage and retrieval systems without permission of Molecular Probes, Inc. All information subject to
`change without notice.
`
`ISBN 0-9710636-0-5 (U.S. dollar version)
`ISBN 0-9710636-1-3 (Euro version)
`ISBN 0-9710636-2-1 (unpriced version) (cid:9)
`
`Printed in the United States of America.
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`www.probes.com
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`3
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`
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`=M~
`
`(cid:149) i
`Handbook of Fluorescent Probes and Research Products
`Customer Service and Ordering Information (cid:9) ...............................................................
`
`
`
`Distributors........................................................................................ iv
`Licensing and Business Development (cid:9) .................................................................... vii
`Howto Use This Book (cid:9) ................................................................................. viii
`Introduction to Fluorescence Techniques (cid:9) .................................................................. 1
`Chapter 1 - Fluorophores and Their Amine-Reactive Derivatives (cid:9) ............................................. 7
`Section 1.1 - (cid:9) Introduction (cid:9) to (cid:9) Amine (cid:9) Modification (cid:9) ....................................................................
`11
`Section 1.2- Kits for (cid:9) Labeling (cid:9) Proteins (cid:9) and (cid:9) Nucleic Acids (cid:9) .............................................................
`14
`Section 1.3 - Alexa (cid:9) Fluor (cid:9) Dyes: (cid:9) Simply the (cid:9) Best (cid:9) .....................................................................
`20
`Section 1.4 - BODIPY (cid:9) Dyes Spanning the Visible Spectrum (cid:9) ............................................................ 36
`Section 1.5 - Fluorescein, Oregon Green and (cid:9) Rhodamine Green (cid:9) Dyes (cid:9) .................................................... 46
`Section 1.6- Dyes with Absorption (cid:9) Maxima above (cid:9) 520 (cid:9) nm (cid:9) ............................................................ 57
`Section 1.7- Fluorophores (cid:9) Excited (cid:9) with (cid:9) UV (cid:9) Light ........ (cid:9) ...................... (cid:9) ......... (cid:9) ............ (cid:9) .................
`66
`Section 1.8 - Reagents for Analysis of Low Molecular Weight Amines ....................................................
`74
`Chapter 2 - Thiol-Reactive Probes (cid:9) ..................................................................... 79
`Section 2.1 - (cid:9) Introduction to Thiol (cid:9) Modification (cid:9) and (cid:9) Detection (cid:9) .......................................................... 81
`Section 2.2 - Thiol-Reactive (cid:9) Probes (cid:9) Excited (cid:9) with Visible (cid:9) Light (cid:9) .......................................................... 84
`Section 2.3 - Thiol-Reactive (cid:9) Probes Excited with (cid:9) Ultraviolet Light (cid:9) ....................................................... 93
`Chapter 3 - Reagents for Modifying Groups Other Than Thiols or Amines
`(cid:9) ..................................... 99
`Section 3.1 -Reagents (cid:9) for (cid:9) Modifying (cid:9) Alcohols (cid:9) .....................................................................
`101
`Section 3.2 - Hydrazines and Aromatic Amines for Modifying Aldehydes and Ketones (cid:9) ...................................... 104
`Section 3.3- Derivatization (cid:9) Reagents for Carboxylic Acids and Glutamine ................................................
`112
`Chapter 4 - Biotin Derivatives and Haptens (cid:9) .............................................................. 119
`Section 4.1 - Introduction to Avidin-Biotin and Antibody-Hapten Techniques
`(cid:9) ............................................. 121
`Section 4.2- Biotinylation (cid:9) and (cid:9) Haptenylation (cid:9) Reagents (cid:9) ..............................................................
`122
`Section 4.3- Biotin (cid:9) and (cid:9) Desthiobiotin (cid:9) Conjugates (cid:9) ..................................................................
`130
`Chapter 5 - Crosslinking and Photoreactive Reagents (cid:9) .................................................... 135
`Section 5.1 - (cid:9) Introduction (cid:9) to (cid:9) Crosslinking (cid:9) Reagents.................................................................
`137
`Section 5.2- Chemical (cid:9) Crosslinking (cid:9) Reagents (cid:9) .....................................................................
`138
`Section 5.3- Photoreactive (cid:9) Crosslinking (cid:9) and (cid:9) Labeling (cid:9) Reagents (cid:9) ....................................................... 144
`Chapter 6 - Ultrasensitive Detection Technology (cid:9) ........................................................ 147
`Section 6.1 - (cid:9) Introduction to (cid:9) Detection (cid:9) Methods (cid:9) ...................................................................
`149
`Section 6.2 - Tyramide Signal Amplification (cid:9) (ISA) Technology (cid:9) ........................................................ 152
`Section 6.3- Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology (cid:9) ........................................ 159
`Section 6.4- Phycobiliproteins (cid:9) .................................................................................
`167
`Section 6.5 - FluoSpheres and TransFluoSpheres Fluorescent Microspheres ..............................................
`175
`Chapter 7 - Antibodies, Avidins, Lectins and Related Products (cid:9) ............................................. 185
`Section 7.1 - A Wide Variety (cid:9) of (cid:9) Protein (cid:9) Conjugates (cid:9) .................................................................
`189
`Section 7.2 - Zenon Technology - Versatile Reagents for Immunolabeling (cid:9) .............................................. 197
`Section 7.3 - (cid:9) Secondary (cid:9) Immunoreagents (cid:9) ........................................................................
`208
`Section 7.4 - Anti-Dye and (cid:9) Anti-Hapten (cid:9) Antibodies .......................................................... (cid:9) ........
`224
`Section 7.5 - Primary Antibodies for Diverse Applications (cid:9) ............................................................ 231
`Section 7.6 - Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices
`(cid:9) ..................... 245
`Section 7.7 - Lectins and (cid:9) Other Carbohydrate-Binding (cid:9) Proteins ........................................................ 258
`
`IJ:-
`
`4
`
`
`
`Chapter 8 - Nucleic Acid Detection and Genomics Technology (cid:9) .............................................. 265
`Section 8.1 - (cid:9) Nucleic (cid:9) Acid (cid:9) Stains (cid:9) ...............................................................................
`269
`Section 8.2- Labeling (cid:9) Oligonucleotides and (cid:9) Nucleic Acids ............................................................ 287
`Section 8.3- Nucleic Acid (cid:9) Detection (cid:9) and (cid:9) Quantitation (cid:9)
`in (cid:9) Solution (cid:9) ......................................................
`300
`Section 8.4 - Nucleic Acid Detection and Quantitation in Electrophoretic Gels and Capillaries
`(cid:9) ................................. 309
`Section 8.5 - Detecting (cid:9) Nucleic Acid (cid:9) Hybridization (cid:9) ..................................................................
`318
`Section 8.6- Nuclear and Chromosome Counterstaining and Nissl Stains ................................................
`338
`Section 8.7- Analysis of DNA Structure, (cid:9) DNA Binding and DNA Damage (cid:9) ................................................
`348
`
`Chapter 9 - Protein Detection and Proteomics Technology (cid:9) ................................................ 353
`Section 9.1 - (cid:9) Introduction (cid:9) to (cid:9) Protein (cid:9) Detection (cid:9) .....................................................................
`355
`Section 9.2- Quantitation and (cid:9) Selective Purification of Proteins in Solution (cid:9) .............................................. 357
`Section 9.3 - Detection of the Total Protein Profile in Gels, on Blots and in Capillary Electrophoresis
`(cid:9) ........................... 365
`Section 9.4- Multiplexed Proteomics for Detection of Specific Proteins in Gels and on Blots
`(cid:9) ................................. 378
`Section 9.5- Reagents for Peptide Analysis, Sequencing and Synthesis (cid:9) .................................................
`394
`
`Chapter 10 - Enzyme Substrates (cid:9) ..................................................................... 397
`Section 10.1 - Introduction to Enzyme Substrates and Their Reference Standards (cid:9) .........................................
`401
`Section 10.2- Detecting (cid:9) Glycosidases (cid:9) ...........................................................................
`406
`Section 10.3 - Detecting Enzymes that Metabolize Phosphates and Polyphosphates ........................................
`420
`Section 10.4- Detecting (cid:9) Peptidases (cid:9) and (cid:9) Proteases (cid:9) .................................................................
`430
`Section 10.5- Substrates for Oxidases, (cid:9) Including Amplex Red (cid:9) Kits ..................................................... 440
`Section 10.6 - Substrates for Miscellaneous Enzymes (cid:9) ...............................................................
`448
`
`Chapter 11 - Probes for Cytoskeletal Proteins .......................................................... 453
`Section 11.1 - (cid:9) Probes (cid:9) for (cid:9) Actin (cid:9) .................................................................................
`455
`Section 11.2- Probes for Tubulin and (cid:9) Other Cytoskeletal (cid:9) Proteins ...................................................... 463
`
`Chapter 12 - Probes for Organelles (cid:9) .................................................................. 409
`Section 12.1 - A (cid:9) Diverse (cid:9) Selection (cid:9) of Organelle (cid:9) Probes (cid:9) ..............................................................
`471
`Section 12.2- Probes (cid:9) for (cid:9) Mitochondria (cid:9) ..........................................................................
`473
`Section 12.3- Probes for Lysosomes, Yeast Vacuoles and Other Acidic Organelles .........................................
`489
`Section 12.4 - Probes for the Endoplasmic Reticulum and Golgi Apparatus...............................................
`496
`
`Chapter 13 - Probes for Lipids and Membranes (cid:9) ......................................................... 503
`Section 13.1 - (cid:9) Introduction (cid:9) to (cid:9) Membrane (cid:9) Probes ...................................................................
`505
`Section 13.2 - Fatty Acid Analogs and (cid:9) Phospholipids (cid:9) ................................................................
`508
`Section 13.3- Sphingolipids, Steroids, Triacylglycerols, Lipopolysaccharides and Related Probes .............................
`524
`Section 13.4- Dial kylcarbocyanine and (cid:9) Dialkylaminostyryl (cid:9) Probes (cid:9) .....................................................
`530
`Section 13.5 - Other Nonpolar and Amphiphilic (cid:9) Probes (cid:9) ..............................................................
`534
`
`Chapter 14 - Fluorescent Tracers of Cell Morphology and Fluid Flow (cid:9) ........................................ 543
`Section 14.1 - (cid:9) Choosing (cid:9) a Tracer (cid:9) ...............................................................................
`547
`Section 14.2- Membrane-Permeant Reactive Tracers for Long-Term Cell Labeling (cid:9) .........................................
`548
`Section 14.3- Polar (cid:9) Tracers (cid:9) ...................................................................................
`554
`Section 14.4- Fluorescent (cid:9) Lipophilic (cid:9) Tracers (cid:9) ......................................................................
`573
`Section 14.5- Fluorescent and (cid:9) Biotinylated (cid:9) Dextrans (cid:9) ................................................................
`581
`Section 14.6- FluoSpheres and TransFluoSpheres Microspheres for Tracing (cid:9) .............................................
`590
`Section 14.7- Protein (cid:9) Conjugates (cid:9) ..............................................................................
`595
`
`Chapter 15 - Assays for Cell Viability, Proliferation and Function
`(cid:9) ........................................... 599
`Section 15.1 - Overview of Probes for Cell Viability, Cell Proliferation and Live-Cell Function
`(cid:9) ................................. 603
`Section 15.2 - Viability and (cid:9) Cytotoxicity Assay (cid:9) Reagents (cid:9) .............................................................
`604
`Section 15.3 - Viability and Cytotoxicity Assay Kits for Diverse Cell Types ................................................
`618
`Section 15.4- Assays for Cell Enumeration, Cell Proliferation and Cell Cycle ..............................................
`631
`Section 15.5 - Assays (cid:9) for (cid:9) Apoptosis (cid:9) .............................................................................
`647
`Section 15.6- Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione
`(cid:9) ................................. 660
`
`www.probes.com
`
`5
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`
`Chapter 16 - Probes for Endocytosis, Receptors and Ion Channels .......................................... 671
`Section 16.1 - Probes for Following Receptor Binding, Endocytosis and Exocytosis (cid:9) ........................................ 673
`Section 16.2- Probes for (cid:9) Neurotransmitter (cid:9) Receptors ............................................................... 693
`Section 16.3 - Probes for (cid:9) Ion (cid:9) Channels (cid:9) and (cid:9) Carriers (cid:9) ................................................................ 701
`
`Chapter 17 - Photoactivatable (Caged) Probes (cid:9) .......................................................... 707
`Section 17.1 - Caging (cid:9) Groups and (cid:9) Their (cid:9) Photolysis (cid:9) ................................................................. 709
`Section 17.2 - Caged (cid:9) Probes for a Variety of Applications (cid:9) ............................................................ 711
`
`Chapter 18 - Probes for Signal Transduction (cid:9) ........................................................... 715
`Section 18.1 - (cid:9) Introduction (cid:9) to (cid:9) Signal (cid:9) Transduction (cid:9) .................................................................. 717
`Section 18.2- Calcium (cid:9) Regulation (cid:9) .............................................................................. 718
`Section 18.3- Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins (cid:9) ............................ 722
`Section 18.4- Probes (cid:9) for (cid:9) Lipid (cid:9) Metabolism (cid:9) and (cid:9) Signaling (cid:9) ............................................................ 734
`
`Chapter 19 - Probes for Reactive Oxygen Species, Including Nitric Oxide (cid:9) .................................... 747
`Section 19.1 - (cid:9) Introduction to (cid:9) Reactive (cid:9) Oxygen (cid:9) Species ......... (cid:9) ............................................... (cid:9) ...... 749
`Section 19.2- Generating and (cid:9) Detecting (cid:9) Reactive Oxygen Species (cid:9) ..................................................... 750
`Section 19.3- Probes (cid:9) for (cid:9) Nitric (cid:9) Oxide (cid:9) Research (cid:9) .................................................................... 762
`Indicators for Ca 21,’ M9 2 , ln 2 (cid:9) and Other Metal Ions ............................................. 767
`Chapter 20 (cid:9)
`Section 20.1 - Introduction to Ca 2 (cid:9) Measurements with Fluorescent Indicators ............................................
`771
`Section 20.2 - Fluorescent Ca (cid:9)
`Indicators (cid:9) Excited with (cid:9) UV Light ....................................................... 776
`Section 20.3 - Fluorescent Ca (cid:9)
`Indicators (cid:9) Excited with (cid:9) Visible (cid:9) Light (cid:9) .................................................... 781
`Section 20.4 - Fluorescent (cid:9) Ca 2+ (cid:9) Indicator (cid:9) Conjugates (cid:9) ................................................................ 795
`Section 20.5 - Aequorin: A (cid:9) Bioluminescent Ca 2 (cid:9)
`Indicator (cid:9) ............................................................ 799
`Section 20.6 - Fluorescent (cid:9) Mg 2 (cid:9)
`Indicators (cid:9) ....................................................................... 802
`Section 20.7- Fluorescent (cid:9) Indicators for Zn 2 (cid:9) and (cid:9) Other Metal (cid:9) Ions (cid:9) .................................................... 805
`Section 20.8- Chelators, Calibration Buffers, (cid:9) lonophores and Cell-Loading Reagents .......................................
`817
`
`Chapter21 (cid:9) - pH Indicators (cid:9) .......................................................................... 827
`Section 21.1 - Overview (cid:9) of (cid:9) pH (cid:9) Indicators (cid:9) ......................................................................... 829
`Section 21.2 - Probes (cid:9) Useful (cid:9) at (cid:9) Near-Neutral (cid:9) pH (cid:9) ................................................................... 830
`Section 21.3 - Probes (cid:9) Useful (cid:9) at (cid:9) Acidic (cid:9) pH (cid:9) ........................................................................ 840
`Section 21.4 - pH (cid:9) Indicator (cid:9) Conjugates (cid:9) .......................................................................... 843
`
`Chapter 22 - Indicators for Na, I(, Cl- and Miscellaneous Ions ............................................ 849
`Section 22.1 - Fluorescent (cid:9) Na (cid:9) and (cid:9) K (cid:9)
`Indicators (cid:9) ................................................................... 851
`Section 22.2 - Detection of Chloride, (cid:9) Phosphate, (cid:9) Nitrite and (cid:9) Other Anions................................................
`856
`
`Probes for Membrane Potential (cid:9) ........................................................... 865
`Chapter 23 (cid:9)
`Section 23.1 - Introduction (cid:9) to (cid:9) Potentiometric (cid:9) Probes ................................................................
`867
`Section 23.2 - Fast-Response (cid:9) Probes (cid:9) ............................................................................ 868
`Section 23.3 - Slow-Response (cid:9) Dyes .................................................... (cid:9) ......................... 871
`
`Chapter 24 - Accessories and Resources (cid:9) .............................................................. 877
`Section 24.1 - Reference Standards and Antifade (cid:9) Reagents (cid:9) ........................................................... 879
`Section 24.2 - Flow Cytometry (cid:9) Reference (cid:9) Standards (cid:9) ................................................................ 888
`Section 24.3 - Accessories for Fluorescence Microscopy and Magnetic Separation .........................................
`893
`Section 24.4 - Photographic Filters for Electrophoretic Gels and (cid:9) Blots ...................................................
`900
`Section 24.5 - Optical (cid:9) Filters for (cid:9) Fluorescence (cid:9) Microscopy (cid:9) ........................................................... 901
`Section 24.6 - Books (cid:9) from (cid:9) Molecular (cid:9) Probes (cid:9) ...................................................................... 906
`
`MasterProduct List (cid:9) ................................................................................. 909
`
`Index ............................................................................................. 951
`
`Trademarks, Patents, Legal Notices and Acknowledgments (cid:9) ................................................. 963
`
`_Ijj_uJ7
`
`6
`
`
`
`1.1
`
`Introduction to Amine Modification
`
`Molecular Probes provides a full spectrum of fluorophores and
`haptens for labeling biopolymers and derivatizing low molecular
`weight molecules. Chapters 1-5 describe the chemical and spec-
`tral properties of the reactive reagents we offer, whereas the
`remainder of this Handbook is primarily devoted to our diverse
`collection of fluorescent probes and their applications in cell
`biology, immunology, biochemistry, biophysics, microbiology,
`molecular biology, genomics, proteomics and neuroscience.
`
`Common Applications for Amine-Reactive Probes
`
`Labeling Biopolymers
`Amine-reactive probes are widely used to modify proteins,
`peptides, ligands, synthetic oligonucleotides and other biomole-
`cules. In contrast to our thiol-reactive reagents (Chapter 2), which
`frequently serve as probes of protein structure and function,
`amine-reactive dyes are most often used to prepare bioconjugates
`for immunochemistry, fluorescence in sitti hybridization (FISH),
`cell tracing, receptor labeling and fluorescent analog cytochemis-
`try.’ In these applications, the stability of the chemical bond
`between the dye and biomolecule is particularly important be-
`cause the conjugate is typically stored and used repeatedly over
`a relatively long period of time. Moreover, these conjugates are
`often subjected to rigorous hybridization and washing steps that
`demand a strong dye(cid:151)biomolecule linkage.
`Our selection of amine-reactive fluorophores for modifying
`biomolecules covers the entire visible and near-infrared spectrum
`(Table 1.1). An up-to-date bibliography is available on our Web
`site for most of our amine-reactive probes. Also available are
`other product-specific bibliographies, as well as keyword search-
`es of the over 44,000 literature references in our extensive bibli-
`ography database. Chapter 1 discusses the properties of Molecu-
`lar Probes’ most important proprietary fluorophores, including
`our premier sets of Alexa Fluor dyes (Section 1.3) and BODIPY
`dyes (Section 1.4), our Oregon Green and Rhodamine Green dyes
`(Section 1.5), the red-fluorescent Rhodamine Red-X and Texas
`Red dyes (Section 1.6) and the UV light(cid:151)excitable Cascade Blue,
`Cascade Yellow, Marina Blue, Pacific Blue and AMCA-X fluoro-
`phores (Section 1.7). Our essentially nonfluorescent QSY dyes
`(Section 1.6, Section 1.8) have strong visible absorption, making
`them excellent acceptors for fluorescence resonance energy
`transfer (FRET, see Section 1.3) applications.
`
`Preparing the Optimal Bioconjugate
`The preferred bioconjugate usually has a high fluorescence
`yield (or, in the case of a haptenylated conjugate, a suitable de-
`gree of labeling) yet retains the critical parameters of the unla-
`beled biomolecule, such as selective binding to a receptor or
`nucleic acid, activation or inhibition of a particular enzyme or the
`ability to incorporate into a biological membrane. Frequently,
`however, conjugates with the highest degree of labeling precipi-
`tate or bind nonspecifically. It may therefore be necessary to have
`a less-than-maximal fluorescence yield to preserve function or
`binding specificity. Although conjugating dyes to biomolecules is
`usually rather easy, preparing the optimal conjugate may require
`extensive experimentation. Thus, for the most critical assays, we
`recommend that researchers consider preparing and optimizing
`
`their own conjugates. We offer a detailed protocol describing how
`to use several of our amine-reactive dyes for labeling biomole-
`cules. The procedure is straightforward and requires no special
`equipment. Following conjugation, it is very important to remove
`as much unconjugated dye as possible, usually by gel filtration,
`dialysis, HPLC or a combination of these techniques. The pres-
`ence of free dye, particularly if it remains chemically reactive,
`can greatly complicate subsequent experiments with the biocon-
`jugate.
`With the exception of the phycobiliproteins (Section 6.4, Table
`6.2), fluorescent microspheres (Section 6.5, Table 6.7), Zenon
`One Labeling Kits (Section 7.2, Table 7.1) and ULYSIS Nucleic
`Acid Labeling Kits (Section 8.2, Table 8.7), virtually all the dyes
`used to prepare Molecular Probes’ fluorescent bioconjugates are
`amine-reactive reagents and almost all are described in this chap-
`ter. We have also developed useful kit formats for labeling pro-
`teins with several of our most important dyes, or alternatively
`with biotin or DSB-X biotin. Table 1.2 and Section 1.2 include a
`complete description of these kits, including our Alexa Fluor and
`FluoReporter Protein Labeling Kits, as well as our new Zenon
`One Labeling Kits (Section 7.2) for the rapid and quantitative
`labeling of mouse IgG 1 antibodies.
`Alternatively, Molecular Probes prepares custom fluorescent
`protein conjugates for research use; contact our Custom and Bulk
`Sales Department for more information. Conjugations with phy-
`cobiliproteins and fluorescent polystyrene microspheres require
`unique procedures that are described in Section 6.4 and Section
`6.5, respectively.
`Molecular Probes also has what are probably the best reagents
`and kits for labeling oligonucleotides and nucleic acids (see
`details in Section 8.2), including:
`
`(cid:149) ARES DNA Labeling Kits (Section 8.2, Table 8.8), which
`permit the indirect labeling of DNA with a wide variety of our
`amine-reactive dyes
`(cid:149) Alexa Fluor Oligonucleotide Amine Labeling Kits (Section
`8.2, Table 8.9) for efficient labeling of 5’-amine-derivatized
`DNA or RNA oligonucleotides with our premiere dyes
`(cid:149) ULYSIS Nucleic Acid Labeling Kits (Section 8.2, Table 8.7),
`which make labeling of nucleic acids as easy as protein labeling
`(cid:149) ChromaTide UTP, ChromaTide OBEA-dCTP and ChromaTide
`dUTP nucleotides labeled with several of our best dyes or with
`biotin (Section 8.2; Table 8.6, Table 8.5), which can be incor-
`porated into nucleic acids by a variety of enzymatic methods 25
`
`In addition, we offer amine-reactive versions of three of our
`SYBR dyes (Section 8.2), which can be conjugated to oligonucle-
`otides, nucleic acids, peptides or proteins that interact with nucle-
`ic acids or affinity matrices. The SYBR dyes remain essentially
`nonfluorescent until complexed to nucleic acids.
`
`Derivallzing Low Molecular Weight Molecules
`Some amine-reactive probes described in this chapter are also
`important reagents for various bioanalytical applications, includ-
`ing amine quantitation, protein and nucleic acid sequencing and
`chromatographic and electrophoretic analysis of low molecular
`weight molecules. Reagents that are particularly useful for deriva-
`
`- IiJs (cid:9)
`
`Section 11
`
`11
`
`7
`
`
`
`tizing low molecular weight amines (cid:9)
`including fluorescamine,
`o-phthaldialdehyde, our ATTO-TAG reagents, NBD chloride and
`dansyl chloride are discussed in Section 1.8. However, many
`of the reactive dyes described in Sections 1.2 to 1.7 can also be
`used as derivatization reagents; likewise, some of the derivatiza-
`tion reagents in Section 1.8 can be utilized for biomolecule conju-
`gation.
`
`Reactivity of Amino Groups
`
`The amine-reactive probes described in this chapter are mostly
`acylating reagents that form carboxamides, sulfonamides, ureas
`or thioureas upon reaction with amines. The kinetics of the reac-
`tion depends on the reactivity and concentration of both the acyl-
`ating reagent and the amine. Of course, buffers that contain free
`amines such as Tris and glycine must be avoided when using any
`amine-reactive probe. Ammonium sulfate that has been used for
`protein precipitation must also be removed before performing dye
`conjugations. In addition, high concentrations of nucleophilic
`thiols should be avoided because they may react with the reagent
`to form an unstable intermediate that could consume the dye.
`Reagents for reductive alkylation of amines (Figure 3.21) are
`described in Chapter 2 and Chapter 3.
`The most significant factors affecting an amine’s reactivity are
`its class and its basicity. Virtually all proteins have lysine resi-
`dues, and most have a free amine at the N-terminus. Aliphatic
`amines such as lysine’s c-amino group are moderately basic and
`reactive with most acylating reagents. However, the concentration
`of the free base form of aliphatic amines below pH 8 is very low;
`thus, the kinetics of acylation reactions of amines by isothiocyan-
`ates, succinimidyl esters and other reagents are strongly pH
`dependent. A pH of 8.5 to 9.5 is usually optimal for modifying
`lysine residues. In contrast, the a-amino group at a protein’s N-
`terminus usually has a pK a of (cid:151)7