Cypridina Luciferase Assay reagent
Product Description: Cypridina (Vargula) Luciferase Assay Reagent:VLAR-1: Assay reagent for Cypridina Luciferase Cypridina (Vargula) luciferase: Cypridina luciferase, (formerly known as Vargula luciferase) from the marine ostracod Vargula Hilgendorfi is a secreted luciferase with an emission max of 460 nm. It is one of the brightest known luciferases with the highest turnover number. Unlike Firefly luciferase, Cypridina lcufierase does nto require ATP and catalyses oxidation of it's unique substrate Cypridina luciferin in a photochemical reaction described below (Figure 1). Cypridina Luciferin is different form colenterazine, the substrate for Renilla, Gaussia and Metridia luciferses. On account of it's unique substrate and bright, secreted luciferase activity Cypridina Luciferase is particularly useful in multiplexed assays involveing Gaussia, Renilla or Firefly luciferses. Secreted CLuc is a very stable protein. Because of this property, the activity measured from the supernatant reflects the amount of protein accumulated up to the time of sampling. Multiple samples can therefore be obtained from the same transfected cells Cypridina luciferase is naturally secreted form cells (Figure 2). Therefore cell lysis is not necessary for measurement of luciferase activity. Figure 2: Intracellular and secreted Cypridina luciferase activity Luciferase activity in cell supernatants and cell lysates of cell transfected with either a plasmid vector expressing secreted vargula luicferase. In cells transfected with the secreted form of modified vargula luciferase, 80% of the activity is sereted into the cell supernatant and only 20% is cell–associated. Stability of Bioluminescent Signal Figure 3: Kinetics of light emission. The stability of the bioluminescent signal of Cypridina Luciferase was assessed using supernatants from HEK 293 cells transiently transfected with the pCMV-VLuc expressionvector. Cypridina Luciferase Assay Protocol: Contents and Storage: Each kit contains the following:
Stability of the undiluted 100X Cypridina substrate is guaranteed for 1 year from the date of purchase. The substrate once diluted should be stored at – 80 ° C and used within 3 months. Preparation of 1X Cypridina substrate and Cypridina assay solution Preparation of 1X Cypridina substrate solution: Dilute the 100X Cypridina luciferase substrate with the appropriate amount of Cypridina substrate dilution buffer. The Assay protocol uses 20 ul of the 1X Cypridina substrate to assay each sample so to assay a 100 samples you would mix 200 ul of the 100X Cypridina substrate with 1.8 ml of the Cypridina substrate dilution buffer. Preparation of the working Cypridina luciferase assay solution: Thaw the VLAR buffer completely at room temperature just before use. Each assay reaction uses a mixture of 20 ul of the Cypridina substrate with 40 ul of the VLAR buffer (Cypridina luciferase assay buffer). For instance if you need to assay 100 samples you should mix 2 ml of 1X Cypridina substrate solution with 4 ml of VLAR buffer just before use. Make sure both solutions are thawed to room temperature prior to mixing. Assay Protocol: Make sure all buffers are at room temperature prior to assay
NOTE: If you need to measure intracellular luciferase activity, lyse cells first using the cell-lysis buffer from Targeting Systems. (catalog no 5X CLR-01)
Luminometer without injectors: Pipette out 5 to 20 ul of Cypridina luciferase sample (supernatant or cell lysate) into each well of a 96-well dish. You may use white (opaque) or black plates or cuvettes. Add 55 ul of the working Cypridina luciferase assay solution (prepared as described above) to each well or cuvette. Mix well and read in the luminometer set for a 2-10 second integration (this can be varied if desired) Luminometer with injectors: Note: Be sure to prepare enough of the working Cypridina luciferase assay solution (prepared as described above) as needed for all samples as well as for priming the injector as suggested by the manufacturer. Protect this solution from light. Set the luminometer with the following parameters: 55 μl injection, 1–2 seconds of delay and 2–10 seconds of integration. Pipet samples (5–20 μl per well) into a 96-well white (opaque) or black plate or cuvettes. Prime the injector with the Cypridina luciferase assay solution and proceed with measurements. References:
Improved luciferases and uses thereof in multiplexed reporter gene assays – application no 61/238146. Custom Reagents: We can provide custom formulations to fit your HTS application. Call our tech support team at 1-866-620-4018 or email us info@targetingsystems.com or targetingsystems@gmail.com Please check out our website www.targetingsystems.net for novel luciferase – based multiplexed assays which enable analysis of up to four promoter activities in the same group of transfected cells. |