HOW TO USE “Prolume Purple II” Cat. #367 Methoxy-COELENTERAZINE-Methoxy patent pending MW 451.52 O O CH3 N N N H O CH3 General Notes: Methoxy-Coelenterazine-Methoxy (Me-O-CTZ-O-Me) called “Prolume Purple II” is a new synthetic analogue of Coelenterazine with two additional methoxy groups. This compound was developed by Nanolight™ Technology to work with Green Renilla Luciferase (GreenRLuc) and Renilla Luciferase 8 (RLuc8) to emit light at ~400 nm. Storage and Shelf Life: It is best stored as completely DRY powder under argon in airtight O-ring plastic tubes at -20°C or for longer storage at -70°C, protected from light. Usage: It is always best to make FRESH SOLUTIONS immediately before luminometer assays or experiments. 1. Dissolve lyophilized Prolume Purple II in NanoFuel Solvent as a 1 mg/ml solution (Ethanol won’t dissolve Me-O-CTZ-O-Me). 2. Use this stock solution to make an aqueous solution in PBS or TBS (e.g. 50 μM for luminometer assays equal to 225.8 μl (1mg/ml) in 10 ml PBS). 3. Store dissolved Prolume Purple II at -80°C, do not store the aqueous working solution (it will oxidize over time). Prolume Purple II has the same stability in aqueous solutions like any other Coelenterazine analogue. NanoLight Technologies division of Prolume Ltd POB 2746 Pinetop, AZ 85935 USA Tel 1-928-367-1200 Fax 1-928-367-1205 www.nanolight.com or www.luciferin.com HOW TO USE “Prolume Purple II” Properties of Nanolight™ Prolume Purple II Cat. #367 A. Comparison of emission spectra of CTZ analogues with Renilla muelleri Luciferase 8 (RLuc8) Emission spectrum of RLuc8 with different CTZ analogues normalized intensity 1 0.8 CTZ native 0.6 Prolume Purple II CTZ400a 0.4 0.2 0 350 400 450 500 550 600 650 wavelength in nm B. Comparison of emission spectra of CTZ analogues with Green Renilla muelleri Luciferase (GreenRLuc) Emission spectrum of Green Luc with different CTZ analogues normalized intensity 1 0.8 native CTZ 0.6 Prolume Purple II 0.4 CTZ400a 0.2 0 350 400 450 500 550 600 650 wavelength in nm NanoLight Technologies division of Prolume Ltd POB 2746 Pinetop, AZ 85935 USA Tel 1-928-367-1200 Fax 1-928-367-1205 www.nanolight.com or www.luciferin.com HOW TO USE “Prolume Purple II” CTZ analogs 60μM each C. Comparison of luminescent intensity between “deep blue C” and “Prolume Purple II” with Green RLuc integrated over 10 sec.: PP-II 400a 0 0.5 1 1.5 RLU/10sec 2 2.5 3 Millions CTZ400a, also known as “deep blue C” and “Prolume Purple II” emit around 400 nm Green RLuc and RLuc8. The luminescent quantum yield of “Prolume Purple II” (PP-II) is around 5-fold higher than CTZ400a. NanoLight Technologies division of Prolume Ltd POB 2746 Pinetop, AZ 85935 USA Tel 1-928-367-1200 Fax 1-928-367-1205 www.nanolight.com or www.luciferin.com