Western blot analysis of secreted Cypridina luciferase was performed using 293T cells stably expressing Gaussia or Cypridina luciferase. Cells were grown overnight in serum free (B27 supplemented) DMEM with or without 10uM Brefeldin A. Equal amounts (75ug) of approximately 10x concentrated cell culture medium from treated or untreated cells, and 10ul of PageRuler Prestained Protein Ladder (Product # 26616) were loaded 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. Cypridina luciferase was detected at ~62kDa using a Cypridina luciferase polyclonal antibody (Product # PA1-182) at a dilution of 1:5,000 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 Dura (Product # 34075). Images were acquired on a Thermo Scientific myECL Imager (Product # 62236).
|Tested species reactivity||Luciferase|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Purified recombinant Cypridina 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)||1:1000-1:10,000|
* 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 PA1-182 detects a ~62 kDa protein in the supernatant of Cypridina luciferase-secreting cells.
PA1-182 can be used for activity-independent verification of Cypridina luciferase expression.
The Cypridina luciferase is a 62kDa protein from the marine ostracod, Cypridina noctiluca. The bioluminescent enzyme is highly secreted into the cell culture media, allowing for live cell monitoring of reporter activity. Light output generated by the luciferase reaction can be correlated to the amount of Cypridina luciferase protein produced and used to determine the activity of the promoter driving Cypridina expression. Cypridina luciferase can be measured individually or in multiplex assays, such as the Cypridina-Firefly Luciferase Dual Assay Kit (Product # 16183). The Cypridina luciferase has been codon optimized for enhanced performance.
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