By Jonathan E. Hempel, Charles H. Williams, Charles C. Hong
This quantity seeks to permit the invention of instruments in chemical biology via delivering readers with a variety of options starting from preliminary chemical genetic screening to focus on id. To effectively spotlight the fundamental elements of the chemical biology device discovery approach, the ebook is organizes into 4 components that concentrate on structures for molecular discovery in in vitro cellular structures, in vivo chemical genetic screening protocols, and strategies used to find useful protein goals. Written within the hugely winning Methods of Molecular Biology sequence layout, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step conveniently reproducible laboratory protocols, and key tips about troubleshooting and keeping off identified pitfalls.
Practical and informative, Chemical Biology: equipment and Protocols seeks to enhance the good fortune cost of the chemical biology box in the course of the dissemination of unique and experiential knowledge.
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Additional resources for Chemical Biology: Methods and Protocols
28 g of N,N′-disuccinimidyl carbonate (10 mM) and 871 μL of N,N-diisopropylethylamine (10 mM) to 500 mL of sieves-dried reagent-grade DMF (for ~100 slides). Prepare prior to use. 9. Photoaffinity PEG linker (Fig. 1a) . 1 Photoaffinity Linker-Coated Slide Preparation Sectional view from line (z) (z) View from top Fig. 3 Teflon® chamber with glass lid and glass slide Photo-Cross-Linked Chemical Array 33 Fig. 4 Synthesis scheme of the proline linker 10. Photoaffinity proline linker (Fig. 1b): See Fig.
Lipofectamine RNAiMax (Invitrogen) (optional, if screening siRNA or miRNA instead of small molecules) (see Note 4). 16. 10 mM Thiazovivin: diluted in DMSO. 17. Ultralow attachment 6-well plates (CoStar). 18. 1 % gelatin. 19. 4 % Paraformaldehyde (PFA) in PBS: add 4 g of PFA to 100 mL of PBS while heating the solution to 65 °C until PFA is completely dissolved. 20 μm filter before use. 20. 1 % Triton X-100. 21. 5 % Triton X-100. 22. 1× PBS (sterile, without magnesium and calcium). 23. Antibodies: MYH6 clone MF20 (supernatant, Hybridoma Bank), α-Actinin (ascites solution, Sigma), PDGFRA directly 46 Sean Spiering et al.
1007/978-1-4939-2269-7_3, © Springer Science+Business Media New York 2015 29 30 Yasumitsu Kondoh et al. Multiple immobilization methods have been reported in earlier studies by several research groups. In general, compounds with reactive functional groups are attached to the surface of glass slides derivatized with certain functional groups by a selective coupling reaction [1, 5, 7, 8]. MacBeath et al.  immobilized small molecules containing free thiols to glass slides coated with maleimide groups by the Michael addition reaction.
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