Bioluminescence: Methods and Protocols by Sung Bae Kim
By Sung Bae Kim
This quantity provides precise laboratory protocols in regards to the 3 significant direction applied sciences luciferases, effective optical probes, and functions to visualizing molecular occasions in dwelling subjects. Bioluminescence: equipment and Protocols, 3rd Edition guides readers via chapters on parts of bioluminescent probes, and fabrication of bioluminescent probes, purposes to dwelling topics and instrumentations. Written within the hugely profitable Methods in Molecular Biology series layout, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and tips about troubleshooting and averting identified pitfalls.
Authoritative and sensible, Bioluminescence: tools and Protocols, 3rd Edition is an invaluable supplement to the 1st and moment variation for brand new and skilled researchers alike.
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Extra resources for Bioluminescence: Methods and Protocols
1 Components for Synthesis of CTZ All solvents and routine reagents for organic synthesis can be purchased from commercial suppliers. 1. 2-Amino-3,5-dibromopyrazine (starting material; store at 5 °C). 2. 0 M in THF (Grignard reagent). 3. Zinc chloride. 4. Tetrakis(triphenylphosphine)palladium(0) (store at −30 °C). 5. Trans-2-phenylvinylboronic acid. 6. Trans-2-(4-methoxyphenyl)vinylboronic acid (Aldrich Chemical). 7. Trans-(2-([1,1′-biphenyl]-4-yl)vinyl)boronic acid (Aldrich Chemical). 8. 0 M Boron tribromide dichloromethane solution (Lewis acid).
Add 500 μL of 80 % ethanol to the column, and centrifuge it for 2 min at 10,000 × g. Discard the flow-through and collection tube. 13. Place the spin column in a fresh 2 mL collection tube, and centrifuge it for 5 min at maximal speed. Discard the flowthrough and collection tube. 14. 5 mL microtube, and add 14 μL of RNase-free water to the column. Centrifuge the column for 1 min at maximal speed to collect the RNA. 2 Reverse Transcription 1. Make the following cocktail to prepare 5′- and 3′-Ready SMART cDNAs.
Precast gel cassette for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) Assay and light-developing reagents 22. Endoplasmic reticulum (ER) tracker (Invitrogen), a live-cell staining reagent emitting fluorescence 23. Renilla luciferase (RLuc) assay system (E2820, Promega), a bioluminescence assay kit 24. Immunostar LD (Warko), a chemiluminescence reagent 25. Native coelenterazine (nCTZ; E2820, Promega) 26. CTZ n, a coelenterazine analogue 27. CTZ i, a coelenterazine analogue 28. CTZ f, a coelenterazine analogue 29.