Elsevier S & T

Bioconjugate Techniques

Hermanson, Greg T.

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Table of contents
  • Cover
  • Bioconjugate Techniquesiii
  • Copyright Pageiv
  • Contentsvii
  • Preface to the Second Editionxxiii
  • Preface to the First Editionxxvi
  • Acknowledgmentsxxviii
  • Health and Safetyxxix
  • Intellectual Propertyxxx
  • PART I: Bioconjugate Chemistry1
  • Chapter 1. Functional Targets3
  • 1. Modification of Amino acids, Peptides, and Proteins3
  • 1.1. Protein Structure and Reactivity4
  • Amino Acids4
  • Nucleophilic Reactions and the pI of Amino Acid Side Chains13
  • Secondary, Tertiary, and Quaternary Structure15
  • Prosthetic Groups, Cofactors, and Post-Translational Modifications19
  • Protecting the Native Conformation and Activity of Proteins21
  • Oxidation of Amino Acids in Proteins and Peptides23
  • Solvent Accessibility of Functional Targets in Proteins29
  • 1.2. Protein Crosslinking Methods32
  • 2. Modification of Sugars, Polysaccharides, and Glycoconjugates35
  • 2.1. Carbohydrate Structure and Functionality36
  • Basic Sugar Structure37
  • Sugar Functional Groups39
  • Polysaccharide and Glycoconjugate Structure44
  • 2.2. Carbohydrate and Glycan Conjugation Methods49
  • 3. Modification of Nucleic Acids and Oligonucleotides50
  • 3.1. Polynucleotide Structure and Functionality51
  • Nucleotide Functional Groups53
  • RNA and DNA Structure62
  • 3.2. Polynucleotide Crosslinking Methods66
  • 4. Creating Specific Functionalities66
  • 4.1. Introduction of Sulfhydryl Residues (Thiolation)67
  • Modification of Amines with 2-Iminothiolane (Traut ’s Reagent)67
  • Modification of Proteins with Cystamine87
  • Modification of Nucleic Acids and Oligonucleotides with Cystamine87
  • Use of Disulfide Reductants87
  • Complete Reduction of Disulfides in Protein Molecules Using DTT90
  • Use of DTT to Cleave Disulfide-Containing Crosslinking Agents91
  • Ellman’s Assay for the Determination of Sulfhydryls100
  • Modification of Amines with SATA71
  • Modification of Amines with SATP74
  • Modification of Amines with SPDP76
  • Modification of Amines with SMPT77
  • Modification of Amines with N -Acetyl Homocysteine Thiolactone80
  • Modification of Amines with SAMSA81
  • Modification of Aldehydes or Ketones with AMBH83
  • Modification of Carboxylates or Phosphates with Cystamine84
  • 4.2. Introduction of Carboxylate Groups101
  • Modification of Amines with Anhydrides102
  • Modification of Sulfhydryls with Iodoacetate109
  • Modification of Sulfhydryls with BMPA111
  • Modification of Hydroxyls with Chloroacetic Acid113
  • 4.3. Introduction of Primary Amine Groups114
  • Modification of Carboxylates with Diamines114
  • Amine Detection Reagents127
  • Modification of Sulfhydryls with N-(Β-Iodoethyl)trifluoroacetamide [Aminoethyl-8]118
  • Modification of Sulfhydryls with Ethylenimine119
  • Modification of Sulfhydryls with 2-Bromoethylamine120
  • Modification of Sulfhydryls with 2-Aminoethyl-2'-aminoethanethiolsulfonate121
  • Modification of Carbohydrates with Diamines122
  • Modification of Alkylphosphates with Diamines124
  • Modification of Aldehydes with Ammonia or Diamines124
  • Introduction of Arylamines on Phenolic Compounds125
  • 4.4. Introduction of Aldehyde Residues129
  • Periodate Oxidation of Glycols and Carbohydrates130
  • Oxidase Modification of Sugar Residues131
  • Modification of Amines with NHS-Aldehydes (SFB and SFPA)132
  • Modification of Amines with Glutaraldehyde134
  • Periodate Oxidation of N-Terminal Serine or Threonine Residues136
  • 4.5. Introduction of Hydrazine or Hydrazide Functionalities139
  • Modification of Aldehydes with Bis-Hydrazide Compounds140
  • Modification of Carboxylates with Bis-Hydrazide Compounds142
  • Modification of Amines with SANH, SHNH, or SHTH143
  • Modification of Alkylphosphates with Bis-Hydrazide Compounds146
  • 4.6. Introduction of Saccharide or Glycan Groups147
  • Modification of Amines with Mono(lactosylamido) mono(succinimidyl)suberate149
  • Modification of Amine or Hydrazide Molecules by Carbohydrates and Glycans150
  • Labeling Glycans with Fluorescent 2-Aminopyridine, 2-Amino Benzamide, or Anthranilic Acid153
  • Synthesis of Glycosylamines for Conjugating Glycans155
  • 5. Blocking Specific Functional Groups156
  • 5.1. Blocking Amine Groups157
  • Sulfo-NHS Acetate157
  • Acetic Anhydride158
  • Citraconic Anhydride159
  • Maleic Anhydride159
  • 5.2. Blocking Sulfhydryl Groups160
  • N-Ethyl Maleimide160
  • Iodoacetate Derivatives161
  • Sodium Tetrathionate161
  • Methyl Methanethiosulfonate163
  • Ellman’s Reagent164
  • Dipyridyl Disulfide Reagents165
  • 5.3. Blocking Aldehyde Groups166
  • Reductive Amination with Tris or Ethanolamine166
  • 5.4. Blocking Carboxylate Groups167
  • Tris or Ethanolamine plus EDC167
  • Chapter 2. The Chemistry of Reactive Groups169
  • 1. Amine Reactions169
  • 1.1. Isothiocyanates170
  • 1.2. Isocyanates170
  • 1.3. Acyl Azides171
  • 1.4. NHS Esters171
  • 1.5. Sulfonyl Chlorides173
  • 1.6. Aldehydes and Glyoxals173
  • 1.7. Epoxides and Oxiranes174
  • 1.8. Carbonates175
  • 1.9. Arylating Agents175
  • 1.10. Imidoesters176
  • 1.11. Carbodiimides176
  • 1.12. Anhydrides178
  • 1.13. Fluorophenyl Esters179
  • 1.14. Hydroxymethyl Phosphine Derivatives180
  • 1.15. Guanidination of Amines181
  • 2. Thiol Reactions182
  • 2.1. Haloacetyl and Alkyl Halide Derivatives182
  • 2.2. Maleimides183
  • 2.3. Aziridines184
  • 2.4. Acryloyl Derivatives184
  • 2.5. Arylating Agents185
  • 2.6. Thiol-Disulfide Exchange Reagents185
  • Pyridyl Disulfides186
  • TNB-Thiol187
  • Disulfide Reductants187
  • 2.7. Vinylsulfone Derivatives188
  • 2.8. Metal-Thiol Dative Bonds188
  • 2.9. Native Chemical Ligation191
  • 2.10. Cisplatin Modification of Methionine and Cysteine192
  • 3. Carboxylate Reactions192
  • 3.1. Diazoalkanes and Diazoacetyl Compounds193
  • 3.2. Carbonyldiimidazole194
  • 3.3. Carbodiimides195
  • 4. Hydroxyl Reactions195
  • 4.1. Epoxides and Oxiranes195
  • 4.2. Carbonyldiimidazole196
  • 4.3. N,N'-Disuccinimidyl Carbonate or N-Hydroxysuccinimidyl Chloroformate196
  • 4.4. Oxidation with Periodate197
  • 4.5. Enzymatic Oxidation198
  • 4.6. Alkyl Halogens198
  • 4.7. Isocyanates199
  • 5. Aldehyde and Ketone Reactions200
  • 5.1. Hydrazine Derivatives200
  • 5.2. Schiff Base Formation200
  • 5.3. Reductive Amination201
  • 5.4. Mannich Condensation201
  • 6. Active Hydrogen Reactions202
  • 6.1. Diazonium Derivatives202
  • 6.2. Mannich Condensation203
  • 6.3. Iodination Reactions203
  • 7. Photo-Chemical Reactions204
  • 7.1. Aryl Azides and Halogenated Aryl Azides204
  • 7.2. Benzophenones205
  • 7.3. Anthraquinones205
  • 7.4. Certain Diazo Compounds207
  • 7.5. Diazirine Derivatives208
  • 7.6. Psoralen Compounds208
  • 8. Cycloaddition Reactions210
  • 8.1. Diels–Alder Reaction210
  • 8.2. Complex Formation with Boronic Acid Derivatives210
  • 8.3. Click Chemistry: Cu[sup(1)]-promoted Azide„Alkyne [3 + 2] Cycloaddition211
  • PART II: Bioconjugate Reagents213
  • Chapter 3. Zero-Length Crosslinkers215
  • 1. Carbodiimides215
  • 1.1. EDC216
  • 1.2. EDC plus Sulfo-NHS219
  • 1.3. CMC223
  • 1.4. DCC224
  • 1.5. DIC226
  • 2. Woodward's Reagent K228
  • 3. N,N'-Carbonyldiimidazole228
  • 4. Schiff Base Formation and Reductive Amination231
  • Chapter 4. Homobifunctional Crosslinkers234
  • 1. Homobifunctional NHS Esters235
  • 1.1. DSP and DTSSP238
  • 1.2. DSS and BS[sup(3)]241
  • 1.3. DST and Sulfo-DST243
  • 1.4. BSOCOES and Sulfo-BSOCOES244
  • 1.5. EGS and Sulfo-EGS246
  • 1.6. DSG248
  • 1.7. DSC249
  • 2. Homobifunctional Imidoesters250
  • 2.1. DMA251
  • 2.2. DMP252
  • 2.3. DMS253
  • 2.4. DTBP254
  • 3. Homobifunctional Sulfhydryl-Reactive Crosslinkers256
  • 3.1. DPDPB257
  • 3.2. BMH258
  • 4. Difluorobenzene Derivatives259
  • 4.1. DFDNB259
  • 4.2. DFDNPS260
  • 5. Homobifunctional Photoreactive Crosslinkers261
  • 5.1. BASED262
  • 6. Homobifunctional Aldehydes262
  • 6.1. Formaldehyde263
  • 6.2. Glutaraldehyde265
  • 7. Bis-epoxides268
  • 7.1. 1,4-Butanediol Diglycidyl Ether269
  • 8. Homobifunctional Hydrazides269
  • 8.1. Adipic Acid Dihydrazide270
  • 8.2. Carbohydrazide271
  • 9. Bis-diazonium Derivatives271
  • 9.1. o-Tolidine, Diazotized272
  • 9.2. Bis-diazotized Benzidine273
  • 10. Bis-alkyl Halides274
  • Chapter 5. Heterobifunctional Crosslinkers276
  • 1. Amine-Reactive and Sulfhydryl-Reactive Crosslinkers277
  • 1.1. SPDP, LC-SPDP, and Sulfo-LC-SPDP278
  • 1.2. SMPT and Sulfo-LC-SMPT281
  • 1.3. SMCC and Sulfo-SMCC283
  • 1.4. MBS and Sulfo-MBS286
  • 1.5. SIAB and Sulfo-SIAB288
  • 1.6. SMPB and Sulfo-SMPB291
  • 1.7. GMBS and Sulfo-GMBS292
  • 1.8. SIAX and SIAXX293
  • 1.9. SIAC and SIACX295
  • 1.10. NPIA296
  • 2. Carbonyl-Reactive and Sulfhydryl-Reactive Crosslinkers297
  • 2.1. MPBH298
  • 2.2. M[sub(2)]C[sub(2)]H299
  • 2.3. PDPH300
  • 3. Amine-Reactive and Photoreactive Crosslinkers302
  • 3.1. NHS-ASA, Sulfo-NHS-ASA, and Sulfo-NHS-LC-ASA305
  • 3.2. SASD306
  • 3.3. HSAB and Sulfo-HSAB308
  • 3.4. SANPAH and Sulfo-SANPAH310
  • 3.5. ANB-NOS312
  • 3.6. SAND312
  • 3.7. SADP and Sulfo-SADP314
  • 3.8. Sulfo-SAPB316
  • 3.9. SAED316
  • 3.10. Sulfo-SAMCA319
  • 3.11. p-Nitrophenyl Diazopyruvate322
  • 3.12. PNP-DTP323
  • 4. Sulfhydryl-Reactive and Photoreactive Crosslinkers324
  • 4.1. ASIB325
  • 4.2. APDP326
  • 4.3. Benzophenone-4-iodoacetamide328
  • 4.4. Benzophenone-4-maleimide330
  • 5. Carbonyl-Reactive and Photoreactive Crosslinkers330
  • 5.1. ABH331
  • 6. Carboxylate-Reactive and Photoreactive Crosslinkers332
  • 6.1. ASBA333
  • 7. Arginine-Reactive and Photoreactive Crosslinkers333
  • 7.1. APG334
  • Chapter 6. Trifunctional Crosslinkers336
  • 1. 4-Azido-2-nitrophenylbiocytin-4-nitrophenyl ester336
  • 2. Sulfo-SBED337
  • 3. MTS-ATF-Biotin and MTS-ATF-LC-Biotin341
  • 4. Hydroxymethyl Phosphine Derivatives342
  • Chapter 7. Dendrimers and Dendrons346
  • 1. Dendrimer Construction346
  • 2. Conjugation to Dendrimers353
  • 2.1. Coupling to Amine-Dendrimers356
  • Modification of Amine-Dendrimers with Sulfo-NHS-LC-SPDP356
  • NHS-PEG-Maleimide Coupling to Amine-Dendrimers359
  • Coupling Glycoproteins to Amine-Dendrimers by Reductive Amination361
  • Blocking of Amines on PAMAM Dendrimers363
  • Preparation of Sugar-Dendrimer Derivatives366
  • Conjugation of Carboxylate Organic Molecules to Amine-Dendrimers371
  • Epoxy Activation of Amine-Dendrimers373
  • Biotinylation of Amine-Dendrimers376
  • Fluorescent Labeling of Amine Dendrimers380
  • 3. Dendrimer-Chelate Derivatives for Imaging Applications383
  • 4. Dendrimer Derivatives as Surface Modification Agents385
  • 5. Dendrimer Fluorescent Quantum Dots389
  • Chapter 8. Cleavable Reagent Systems391
  • 1. Cleavage of Disulfides by Reduction392
  • 2. Periodate-Cleavable Glycols393
  • 3. Dithionite-Cleavable Bonds394
  • 4. Hydroxylamine Cleavable Esters394
  • 5. Base Labile Sulfones395
  • Chapter 9. Fluorescent Probes396
  • 1. Fluorescein Derivatives400
  • Amine-Reactive Fluorescein Derivatives401
  • Sulfhydryl-Reactive Fluorescein Derivatives406
  • Aldehyde/Ketone and Cytosine-Reactive Fluorescein Derivatives412
  • 2. Rhodamine Derivatives415
  • Amine-Reactive Rhodamine Derivatives416
  • Sulfhydryl-Reactive Rhodamine Derivatives425
  • Aldehyde/Ketone and Cytosine-Reactive Rhodamine Derivatives427
  • 3. Coumarin Derivatives430
  • Amine-Reactive Coumarin Derivatives431
  • Sulfhydryl-Reactive Coumarin Derivatives434
  • Aldehyde- and Ketone-Reactive Coumarin Derivatives438
  • 4. BODIPY Derivatives440
  • Amine-Reactive BODIPY Derivatives441
  • Aldehyde/Ketone-Reactive BODIPY Derivatives444
  • Sulfhydryl-Reactive BODIPY Derivatives449
  • 5. Cascade Blue Derivatives453
  • Amine-Reactive: Cascade Blue Acetyl Azide453
  • Carboxylate-Reactive: Cascade Blue Cadaverine and Cascade Blue Ethylenediamine455
  • Aldehyde/Ketone-Reactive: Cascade Blue Hydrazide456
  • 6. Lucifer Yellow Derivatives457
  • Sulfhydryl-Reactive: Lucifer Yellow Iodoacetamide458
  • Aldehyde/Ketone-Reactive: Lucifer Yellow CH459
  • 7. Phycobiliprotein Derivatives461
  • 8. Cyanine Dye Derivatives464
  • Amine-reactive Cyanine Dyes467
  • Thiol-reactive Cyanine Dyes470
  • Carbonyl-reactive Cyanine Dyes472
  • 9. Lanthanide Chelates for Time-resolved Fluorescence474
  • 10. Quantum Dot Nanocrystals485
  • Properties of Quantum Dots485
  • Conjugation to QDs493
  • Conjugation of Proteins to QDs Using EDC494
  • Conjugation to QDs Using sulfo-SMCC496
  • Chapter 10. Bifunctional Chelating Agents and Radioimmunoconjugates498
  • 1. DTPA499
  • 2. DOTA, NOTA, and TETA500
  • 3. DTTA501
  • 4. DFA502
  • 5. Use of Thiolation Reagents for Direct Labeling to Sulfhydryl Groups503
  • 6. FeBABE505
  • Chapter 11. Biotinylation Reagents506
  • 1. Amine-Reactive Biotinylation Agents507
  • 1.1. D-Biotin and Biocytin508
  • 1.2. NHS-Biotin and Sulfo-NHS-Biotin510
  • 1.3. NHS-LC-Biotin and Sulfo-NHS-LC-Biotin512
  • 1.4. NHS-Iminobiotin515
  • 1.5. Sulfo-NHS-SS-Biotin517
  • 2. Sulfhydryl-Reactive Biotinylation Agents520
  • 2.1. Biotin-BMCC520
  • 2.2. Biotin-HPDP522
  • 2.3. Iodoacetyl-LC-Biotin524
  • 3. Carbonyl- or Carboxyl-Reactive Biotinylation Agents525
  • 3.1. Biotin-Hydrazide and Biotin-LC-Hydrazide526
  • 3.2. Biocytin Hydrazide528
  • 3.3. 5-(Biotinamido)pentylamine529
  • 4. Photoreactive Biotinylation Agents530
  • 4.1. Photobiotin531
  • 4.2. Psoralen-PEO[sub(3)]-Biotin533
  • 5. Active Hydrogen-Reactive: p-Aminobenzoyl Biocytin, Diazotized534
  • 6. Glycan Biotinylation Reagents537
  • 6.1. Biotinylated Aminopyridine538
  • 6.2. Biotinyl-L-3-(2-naphthyl)-alanine hydrazide541
  • 6.3. Biotin-PEG-Phosphine543
  • Chapter 12. Iodination Reagents546
  • 1. Chloramine-T548
  • 2. Iodobeads550
  • 3. Iodogen553
  • 4. Lactoperoxidase-Catalyzed Iodination555
  • 5. Iodinatable Modification and Crosslinking Agents556
  • 5.1. Bolton–Hunter Reagent557
  • 5.2. Iodinatable Bifunctional Crosslinking Agents560
  • Chapter 13. Silane Coupling Agents562
  • 1. Silane Reaction Strategies565
  • 1.1. Aqueous/Organic Solvent Deposition566
  • 1.2. Aqueous Deposition566
  • 1.3. Organic Solvent Deposition567
  • 1.4. Vapor Phase Deposition567
  • 2. Functional Silane Compounds568
  • 2.1. 3-Aminopropyltriethoxysilane and 3-Aminopropyltrimethoxysilane568
  • 2.2. Carboxyethylsilanetriol573
  • 2.3. N-(Trimethoxysilylpropyl)ethylenediamine triacetic acid575
  • 2.4. 3-Glycidoxypropyltrimethoxysilane and 3-Glycidoxypropyltriethoxysilane577
  • 2.5. Isocyanatopropyltriethoxysilane579
  • Chapter 14. Microparticles and Nanoparticles582
  • 1. Particle Types582
  • 2. Particle Characteristics and Stability584
  • 3. Particle Concentration588
  • 4. Polymeric Microspheres and Nanospheres588
  • 4.1. Passive Adsorption590
  • 4.2. Covalent Coupling to Polymeric Particles594
  • 4.3. Coupling to Carboxylate Particles595
  • 4.4. Coupling to Amine Particles599
  • 4.5. Coupling to Amine Particles Using Crosslinking Agents599
  • 4.6.Glutaraldehyde601
  • 4.7. SPDP Coupling to Amine Particles602
  • 4.8. NHS-PEG[sub(n)]-Maleimide Coupling to Amine Particles604
  • 4.9. Coupling to Hydroxyl Particles606
  • 4.10. Coupling to Hydrazide Particles613
  • 4.11. Coupling to Epoxy Particles615
  • 4.12. Coupling to Aldehyde Particles617
  • 5. Silica Particles618
  • 5.1. Fluorescent Silica Particles620
  • 5.2. Silane Functionalization of Silica Particles625
  • Chapter 15. Buckyballs, Fullerenes, and Carbon Nanotubes627
  • 1. Buckyballs and Fullerenes627
  • 1.1. Properties of Fullerenes627
  • 1.2. Modification of Fullerenes629
  • 2. Carbon Nanotubes638
  • 2.1. Nanotube Properties638
  • 2.2. Nanotube Functionalization640
  • 2.3. Detergent or Lipid Modification of Carbon Nanotubes640
  • 2.4. Pyrene Modification of Carbon Nanotubes644
  • 2.5. Modification of Carbon Nanotubes by Cycloaddition645
  • Chapter 16. Mass Tags and Isotope Tags649
  • 1. ICAT Reagents651
  • 2. ECAT Reagents657
  • 3. Isobaric Tags659
  • Chapter 17. Chemoselective Ligation: Bioorthogonal Reagents666
  • 1. Diels–Alder Reagent Pairs667
  • 2. Hydrazine–Aldehyde Reagent Pairs669
  • Protocol for Modification of Amine-Oligo with SANH or SFB674
  • Protocol for Modification of Protein or Antibody with SANH or SFB675
  • Conjugation Using the Aldehyde/Hydrazine Reaction675
  • 3. Boronic Acid–Salicylhydroxamate Reagent Pairs676
  • 4. Click Chemistry: Cu(I)-Promoted Azide–Alkyne [3 + 2] Cycloaddition680
  • 5. Staudinger Ligation690
  • 6. Native Chemical Ligation697
  • 6.1. Expressed Protein Ligation and Inteins701
  • Chapter 18. Discrete PEG Reagents707
  • 1. Homobifunctional PEG Crosslinkers711
  • 1.1. Bis-NHS Ester PEG Compounds711
  • 1.2. Bis-Maleimide–PEG Compounds714
  • BM(PEG)[sub(2)], BM(PEG)[sub(3)], and Bis-MAL–dPEG[sub(3)]714
  • 2. Heterobifunctional PEG Reagents718
  • 2.1. Maleimide–PEG[sub(n)]–NHS Ester Compounds718
  • 2.2. NHS-PEG[sub(n)]-Azide/Alkyne Compounds for Chemoselective Ligation722
  • 3. Biotinylation Reagents Containing Discrete PEG Linkers726
  • 3.1. NHS–PEG[sub(n)]–Biotin Compounds727
  • 3.2. NHS-Chromogenic-PEG[sub(3)]-Biotin730
  • 3.3. Maleimide-PEG[sub(n)]-Biotin Compounds732
  • 3.4. Hydrazide-PEG[sub(4)]-Biotin733
  • 3.5. Biotin-PEG[sub(n)]-Amine Compounds736
  • 3.6. Biotin–PEG[sub(3)]–Benzophenone739
  • 4. Discrete PEG Modification Reagents739
  • PART III: Bioconjugate Applications743
  • Chapter 19. Preparation of Hapten–Carrier Immunogen Conjugates745
  • 1. The Basis of Immunity745
  • 2. Types of Immunogen Carriers747
  • 2.1. Protein Carriers748
  • KLH748
  • BSA and cBSA749
  • Thyroglobulin and OVA751
  • Tetanus and Diphtheria Toxoids753
  • 2.2. Liposome Carriers753
  • 2.3. Synthetic Carriers754
  • 3. Carbodiimide-Mediated Hapten–Carrier Conjugation755
  • 4. NHS Ester-Mediated Hapten–Carrier Conjugation763
  • 5. NHS Ester-Maleimide Heterobifunctional Crosslinker-Mediated Hapten–Carrier Conjugation766
  • 6. Active-Hydrogen-Mediated Hapten–Carrier Conjugation773
  • 6.1. Diazonium Conjugation773
  • 6.2. Mannich Condensation776
  • 7. Glutaraldehyde-Mediated Hapten–Carrier Conjugation779
  • 8. Reductive Amination-Mediated Hapten–Carrier Conjugation781
  • Chapter 20. Antibody Modification and Conjugation783
  • 1. Preparation of Antibody–Enzyme Conjugates787
  • 1.1. NHS Ester–Maleimide-Mediated Conjugation788
  • Activation of Enzymes with NHS Ester–Maleimide Crosslinkers789
  • Conjugation with Reduced Antibodies790
  • Conjugation with 2-Iminothiolane-Modified Antibodies793
  • Conjugation with SATA-Modified Antibodies795
  • 1.2. Glutaraldehyde-Mediated Conjugation797
  • One-Step Glutaraldehyde Protocol798
  • Two-Step Glutaraldehyde Protocol800
  • 1.3. Reductive-Amination-Mediated Conjugation800
  • Activation of Enzymes with Sodium Periodate802
  • Activation of Antibodies with Sodium Periodate803
  • Conjugation of Periodate-Oxidized HRP to Antibodies by Reductive Amination804
  • Conjugation of Periodate-Oxidized Antibodies with Amine or Hydrazide Derivatives805
  • 1.4. Conjugation Using Antibody Fragments807
  • Preparation of F(ab')[sub(2)] Fragments Using Pepsin807
  • Preparation of Fab Fragments Using Papain808
  • 1.5. Removal of Unconjugated Enzyme from Antibody–Enzyme Conjugates812
  • Immunoaffinity Chromatography813
  • Nickel-Chelate Affinity Chromatography814
  • 2. Preparation of Labeled Antibodies816
  • 2.1. Fluorescently Labeled Antibodies817
  • 2.2. Radiolabeled Antibodies819
  • 2.3. Biotinylated Antibodies821
  • Chapter 21. Immunotoxin Conjugation Techniques824
  • 1. Properties and Use of Immunotoxin Conjugates827
  • 2. Preparation of Immunotoxin Conjugates829
  • 2.1. Preparation of Immunotoxin Conjugates via Disulfide Exchange Reactions833
  • Pyridyl Disulfide Reagents834
  • SPDP834
  • SMPT841
  • 3-(2-Pyridyldithio) Propionate843
  • Use of Cystamine, Ellman’s Reagent, or S-Sulfonates844
  • 2.2. Preparation of Immunotoxin Conjugates via Amine- and Sulfhydryl-Reactive Heterobifunctional Cro847
  • SIAB847
  • SMCC850
  • MBS852
  • SMPB854
  • 2.3. Preparation of Immunotoxin Conjugates via Reductive Amination855
  • Periodate Oxidation of Glycoproteins Followed by Reductive Conjugation855
  • Periodate-Oxidized Dextran as Crosslinking Agent857
  • Chapter 22. Preparation of Liposome Conjugates and Derivatives858
  • 1. Properties and Use of Liposomes858
  • 1.1. Liposome Morphology858
  • 1.2. Preparation of Liposomes861
  • 1.3. Chemical Constituents of Liposomes863
  • 1.4. Functional Groups of Phospholipids869
  • 2. Derivatization and Activation of Lipid Components869
  • 2.1. Periodate Oxidation of Liposome Components870
  • 2.2. Activation of PE Residues with Heterobifunctional Crosslinkers871
  • 3. Use of Glycolipids and Lectins to Effect Specific Conjugations877
  • 4. Antigen or Hapten Conjugation to Liposomes879
  • 5. Preparation of Antibody–Liposome Conjugates881
  • 6. Preparation of Biotinylated or Avidin-Conjugated Liposomes883
  • 7. Conjugation of Proteins to Liposomes885
  • 7.1. Coupling via the NHS Ester of Palmitic Acid886
  • 7.2. Coupling via Biotinylated PE Lipid Derivatives888
  • 7.3. Conjugation via Carbodiimide Coupling to PE Lipid Derivatives888
  • 7.4. Conjugation via Glutaraldehyde Coupling to PE Lipid Derivatives890
  • 7.5. Conjugation via DMS Crosslinking to PE Lipid Derivatives892
  • 7.6. Coupling via Periodate Oxidation Followed by Reductive Amination893
  • 7.7. Conjugation via SPDP-Modified PE Lipid Derivatives894
  • 7.8. Conjugation via SMPB-Modified PE Lipid Derivatives895
  • 7.9. Conjugation via SMCC-Modified PE Lipid Derivatives896
  • 7.10. Conjugation via Iodoacetate-Modified PE Lipid Derivatives897
  • Chapter 23. Avidin–Biotin Systems900
  • 1. The Avidin–Biotin Interaction900
  • 2. Use of (Strept)avidin–Biotin Interactions in Assay Systems902
  • 3. Preparation of (Strept)avidin Conjugates905
  • 3.1. NHS Ester–Maleimide-Mediated Conjugation Protocols906
  • 3.2. Conjugation Using Periodate Oxidation/Reductive Amination910
  • 3.3. Glutaraldehyde Conjugation Protocol913
  • 4. Preparation of Fluorescently Labeled (Strept)avidin914
  • 4.1. Modification with FITC915
  • 4.2. Modification with Lissamine Rhodamine B Sulfonyl Chloride916
  • 4.3. Modification with AMCA–NHS917
  • 4.4. Conjugation with Phycobiliproteins918
  • 5. Preparation of Hydrazide-Activated (Strept)avidin919
  • 6. Biotinylation Techniques920
  • 7. Determination of the Level of Biotinylation921
  • Chapter 24. Preparation of Colloidal Gold-Labeled Proteins924
  • 1. Properties and Use of Gold Conjugates924
  • 2. Preparation of Mono-Disperse Gold Suspensions for Protein Labeling928
  • 2.1. Preparation of 2 nm Gold Particle Sols928
  • 2.2. Preparation of 5 nm Gold Particle Sols928
  • 2.3. Preparation of 12 nm Gold Particle Sols929
  • 2.4. Preparation of 30 nm Gold Particle Sols929
  • 3. Preparation of Protein A–Gold Complexes930
  • 4. Preparation of Antibody–Gold Complexes931
  • 5. Preparation of Lectin–Gold Complexes932
  • 6. Preparation of (Strept)avidin–Gold Complexes934
  • Chapter 25. Modification with Synthetic Polymers936
  • 1. Protein Modification with Activated Polyethylene Glycols937
  • 1.1. Trichloro-s-triazine Activation and Coupling938
  • 1.2. NHS Ester and NHS Carbonate Activation and Coupling940
  • 1.3. Carbodiimide Coupling of Carboxylate–PEG Derivatives945
  • 1.4. CDI Activation and Coupling946
  • 1.5. Miscellaneous Coupling Reactions948
  • 2. Protein Modification with Activated Dextrans951
  • 2.1. Polyaldehyde Activation and Coupling952
  • 2.2. Carboxyl, Amine, and Hydrazide Derivatives954
  • 2.3. Epoxy Activation and Coupling956
  • 2.4. Sulfhydryl-Reactive Derivatives960
  • Chapter 26. Enzyme Modification and Conjugation961
  • 1. Properties of Common Enzymes961
  • 1.1. Horseradish Peroxidase961
  • 1.2. Alkaline Phosphatase963
  • 1.3. Β-Galactosidase964
  • 1.4. Glucose Oxidase965
  • 2. Preparation of Activated Enzymes for Conjugation966
  • 2.1. Glutaraldehyde-Activated Enzymes966
  • 2.2. Periodate Oxidation Techniques966
  • 2.3. SMCC-Activated Enzymes967
  • 2.4. Hydrazide-Activated Enzymes967
  • 2.5. SPDP-Activated Enzymes968
  • 3. Preparation of Biotinylated Enzymes968
  • Chapter 27. Nucleic Acid and Oligonucleotide Modification and Conjugation969
  • 1. Enzymatic Labeling of DNA970
  • 2. Chemical Modification of Nucleic Acids and Oligonucleotides973
  • 2.1. Diamine or Bis-Hydrazide Modification of DNA974
  • Conjugation via Bisulfite Activation of Cytosine974
  • Conjugation via Bromine Activation of Thymine, Guanine, and Cytosine976
  • Conjugation via Carbodiimide Reaction with the 5' Phosphate of DNA (Phosphoramidate Formation)978
  • 2.2. Sulfhydryl Modification of DNA980
  • Cystamine Modification of 5' Phosphate Groups Using EDC981
  • SPDP Modification of Amines on Nucleotides982
  • SATA Modification of Amines on Nucleotides984
  • 2.3. Biotin Labeling of DNA985
  • Biotin-LC-dUTP985
  • Photobiotin Modification of DNA987
  • Reaction of NHS-LC-Biotin with Diamine-Modified DNA Probes987
  • Biotin–Diazonium Modification of DNA989
  • Reaction of Biotin–BMCC with Sulfhydryl-Modified DNA990
  • Biotin–Hydrazide Modification of Bisulfite-Activated Cytosine Groups990
  • 2.4. Enzyme Conjugation to DNA992
  • Alkaline Phosphatase Conjugation to Cystamine-Modified DNA Using Amine- and Sulfhydryl-Reactive Hete993
  • Alkaline Phosphatase Conjugation to Diamine-Modified DNA Using DSS994
  • Enzyme Conjugation to Diamine-Modified DNA Using PDITC996
  • Conjugation of SFB-Modified Alkaline Phosphatase to Bis-Hydrazide-Modified Oligonucleotides998
  • 2.5. Fluorescent Labeling of DNA998
  • Conjugation of Amine-Reactive Fluorescent Probes to Diamine-Modified DNA1001
  • Conjugation of Sulfhydryl-Reactive Fluorescent Probes to Sulfhydryl-Modified DNA1002
  • Chapter 28. Bioconjugation in the Study of Protein Interactions1003
  • 1. Homobifunctional Crosslinking Agents1006
  • 1.1. DSS and BS[sup(3)]1007
  • 1.2. Heavy Atom, Deuterated Crosslinking Agents1008
  • 1.3. Formaldehyde1010
  • 1.4. Protein Interaction Reporters1011
  • 2. Use of Photoreactive Crosslinkers to Study Protein Interactions1016
  • 2.1. Sulfo-SAND, SANPAH, and Sulfo-SANPAH1016
  • 2.2. Sulfo-SFAD1018
  • 3. Trifunctional Label Transfer Reagents1020
  • 3.1. Sulfo-SBED1021
  • 3.2. MTS-ATF-Biotin and MTS-ATF-LC-Biotin1028
  • 4. Metal Chelates in the Study of Protein Interactions1032
  • 4.1. FeBABE for Protein Mapping Studies1032
  • 4.2. Ru(II)bpy[sub(3)][sup(2+)] for Light-Triggered Zero-Length Crosslinking of Protein Interactions1037
  • References1041
  • Index1133
  • A1133
  • B1140
  • C1144
  • D1150
  • E1155
  • F1158
  • G1160
  • H1163
  • I1167
  • J1170
  • K1171
  • L1172
  • M1174
  • N1177
  • O1179
  • P1180
  • Q1187
  • R1187
  • S1189
  • T1197
  • U1200
  • V1201
  • W1201
  • X1202
  • Y1202
  • Z1202
Book details
  • Vendor Elsevier S & T
  • SKU 9780123705013
  • ISBN-13 9780080568720
  • Author Hermanson, Greg T.
  • Edition 2nd
  • Category Science
  • Subject Research & Methodology

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Bioconjugate Techniques, 2nd Edition, is the essential guide to the modification and cross linking of biomolecules for use in research, diagnostics, and therapeutics. It provides highly detailed information on the chemistry, reagent systems, and practical applications for creating labeled or conjugate molecules. It also describes dozens of reactions with details on hundreds of commercially available reagents and the use of these reagents for modifying or cross linking peptides and proteins, sugars and polysaccharides, nucleic acids and oligonucleotides, lipids, and synthetic polymers.

*A one-stop source for proven methods and protocols for synthesizing bioconjugates in the lab
*Step-by-step presentation makes the book an ideal source for researchers who are less familiar with the synthesis of bioconjugates
*More than 600 figures that visually describe the complex reactions associated with the synthesis of bioconjugates
*Includes entirely new chapters on the latest areas in the field of bioconjugation as follows:
Microparticles and nanoparticles
Silane coupling agents
Dendrimers and dendrons
Chemoselective ligation
Quantum dots
Lanthanide chelates
Cyanine dyes
Discrete PEG compounds
Buckyballs,fullerenes, and carbon nanotubes
Mass tags and isotope tags
Bioconjugation in the study of protein interactions