Western blot analysis of TurboLuc luciferase was performed by loading the indicated amounts of recombinant TurboLuc whole cell lysates, and 10ul of PageRuler Plus Prestained Protein Ladder (Product # 26619) onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for 1 hour at room temperature. TurboLuc luciferase was detected at 16kDa using a TurboLuc luciferase polyclonal antibody (Product # PA1-178) at a dilution of 1:1000 in StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). Images were acquired on a Thermo Scientific myECL Imager (Product # 62236). Note: Additional weak bands marked with asterisks at ~32kDa and ~48kDa are also detected, likely representing undenatured forms of recombinant TurboLuc.
|Tested species reactivity||Luciferase|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Purified recombinant TurboLuc luciferase expressed in E. coli.|
|Storage buffer||PBS with 1mg/ml BSA, 30% glycerol|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Western blot analysis of PA1-178 detects an ~16-18 kDa protein in Tluc expressing cells.
PA1-178 can be used for activity-independent verification of Tluc expression in qIP assays.
TurboLuc™ (Tluc) is a novel intracellular luciferase derived from the marine copepod Metridia luciferase family. The Tluc gene was developed using directed mutagenesis for optimal performance and codon optimized for mammalian cell expression. Tluc is an ATP-independent enzyme that utilizes the substrate coelenterazine to produce intense bioluminescence light. Tluc is smaller and brighter than other commonly used luciferases, including firefly and Renilla. The intense bioluminescence greatly enhances sensitivity of Tluc luciferase-based assays, enabling detection of very minute amounts of luciferase (signal) in very sensitive assays such as protein interaction qIP assays (Prod # 82032, 82033, 82035, 82036).
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