Thermo Scientific Pierce Crosslinking Technical Handbook

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Crosslinker Reactivities Diazirines The succinimidyl-ester diazirine (SDA) reagents are a new class of crosslinkers that combine proven amine-reactive chemistry with an efficient diazirine-based photochemistry for photo-crosslinking to nearly any other functional group. Diazirine-based photocrosslinkers have better photostability than phenyl azide-based photocrosslinkers and are easily activated with long-wave UV light (330-370 nm). The SDA crosslinkers include six compounds differing in spacer arm lengths, ability to cleave the crosslinked proteins, and presence or absence of a charged group for differential membrane permeability. These SDA reagents extend the efficiency and range of interactions that can be explored by this approach. Photoactivation of diazirine with long-wave UV light (330-370 nm) creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side-chain or peptide backbone at distances corresponding to the spacer arm lengths. The NHS-ester diazirine derivatives (SDA, LC-SDA and SDAD) lack a charged group and are membrane-permeable. This property makes them ideal for intracellular and intramembrane conjugations. By contrast, Sulfo-SDA, Sulfo-LC-SDA and Sulfo-SDAD contain negatively charged sulfonate groups that improve their water solubility and reduce cell membrane permeability, enabling their use for extracellular protein crosslinking. SDAD and Sulfo-SDAD also have a disulfide bond within the spacer that can be cleaved with reducing agents. Thermo Scientific Pierce Diazirine-based Photo-crosslinkers. Reagent Product # Reactive Groups SDA 26167 NHS, Diazirine LC-SDC 26168 NHS, Diazirine SDAD 26169 NHS, Diazirine Sulfo-SDA 26173 Sulfo-NHS, Diazirine Sulfo-LC-SDA 26174 Sulfo-NHS, Diazirine Sulfo-SDAD 26175 Sulfo-NHS, Diazirine Protein 1 Protein 1 NH 2 O N N O N H N N 2 UV 350 nm O N N O O Protein 2 Protein 1 H N O H Protein 2 Figure 15. Mechanism of NHS-ester diazirine crosslinking. N-hydroxysuccinimide (NHS) esters react efficiently with primary amine groups (–NH 2 ) in pH 7-9 buffers to form stable amide bonds upon release of NHS. Photoactivation of diazirines with long-wave UV light (330-370 nm) creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths. 22 For more information, or to download product instructions, visit www.thermo.com/pierce
Books and Free Technical Handbooks! Bioconjugate Techniques, 2nd Edition Bioconjugate Techniques is the best single source of comprehensive knowledge and techniques required to design and synthesize any bioconjugate. This is a must-have resource when searching for methods that will facilitate the conjugation of molecules to new reagents or for novel applications. As the field of bioconjugation continues to grow, researchers in academia and industry look to link two or more molecules to form novel complexes with uniquely combined properties of the individual components. Greg Hermanson, a recognized bioconjugation technology expert, has written a second edition of the Bioconjugate Techniques book. It provides a complete update and major expansion of the original work which has been established as the definitive reference in the field. This 1202-page book is an extremely valuable source of information with easy-to-understand strategies, 850 figures and illustrations and up-to-date references. It comprehensively covers every reagent system for bioconjugation in research, diagnostics, and therapeutic development. It also discusses the latest advances in bioconjugation methods applied to nanotechnology and proteinprotein interactions. Product # 20036 Hermanson, G.T. (2008). Bioconjugate Techniques, 2nd ed., Elsevier Inc., 1202 pages. Sorry, books are nonreturnable. Thermo Scientific Avidin-Biotin Technical Handbook Chemistry of Protein Conjugation and Cross-Linking An excellent crosslinking reagent reference for the expert and the beginner. Details the critical aspects of protein crosslinking and conjugate preparation and covers the full spectrum from protein-reactive groups to proteinnucleic acid crosslinking. Product # 15010 Wong, S.S., Ph.D., Published by CRC Press, Inc., 1991, 340 pages, Hardcover. Sorry, books are nonreturnable. Avidin-Biotin Products This reference guide brings together everything needed to biotinylate cellsurface proteins, purify a biotinylated target, detect a biotinylated antibody and perform many other applications. It includes dozens of references along with protocols, troubleshooting tips, selection guides and a complete listing of available tools. Because the Avidin-Biotin system can be used in so many ways, you’ll want to keep this handbook close at hand! Product # 1601676 / Free! To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 23
Page 1 and 2: Thermo Scientific Pierce Crosslinki
Page 3 and 4: Introduction What is crosslinking C
Page 5 and 6: Crosslinking Applications Crosslink
Page 7 and 8: Solid-phase immobilization Proteins
Page 9 and 10: O NH Biotin HN S SO 3 - Cleavable D
Page 11 and 12: Sulfo-SBED Reagent Thermo Scientifi
Page 13 and 14: Figure 3. Applications of Sulfo-SBE
Page 15 and 16: Structure determination with heavy/
Page 17 and 18: Bioconjugate Toolkit The next gener
Page 19 and 20: Crosslinker Reactivities Amine-reac
Page 21 and 22: Pyridyl disulfides Pyridyl disulfid
Page 23: Phenyl Azide UV Light Nitrene Forma
Page 27 and 28: - SH Sulfhydryls X Carbohydrates No
Page 29 and 30: - SH Sulfhydryls X Carbohydrates No
Page 31 and 32: - SH Sulfhydryls Carbohydrates Nons
Page 33 and 34: Appendix I Structures Product # Pro
Page 47 and 48: Appendix III - Glossary of Crosslin

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