pNL3.2.NF-κB-RE BioVector®哺乳动物NanoLuc荧光融合表达载体-BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

pNL3.2.NF-κB-RE BioVector®哺乳动物NanoLuc荧光融合表达载体-BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

The pNL3.2.NF-κB-RE Vector contains 5 copies of an NF-κB response element (NF-κB-RE) that drives transcription of a destabilized form of NanoLuc luciferase, an engineered 23.3kDa luciferase fusion protein. The NlucP reporter consists of NanoLuc luciferase with a C-terminal fusion to PEST, a protein destabilization domain, which responds more quickly and with greater magnitude to changes in transcriptional activity than unmodified NanoLuc® luciferase. The NlucP gene is codon optimized for expression in mammalian cells, and all pNL vectors and Nluc genes have minimal consensus transcription factor-binding sites to reduce anomalous expression. The vector contains an ampicillinresistance gene for selection in E. coli and a selectable marker for hygromycin resistance in mammalian cells.

Concentration: 1µg/µl.GenBank® Accession Number: JQ513377.Storage Buffer: pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] Vector is supplied in 10mM Tris-HCl (pH 7.4), 1mM EDTA.Storage Conditions: See the product information label for storage recommendations and expiration date.

pNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] Vector Features Listand Circle MapNF-κB response elements 33–84Minimal promoter 117–147NlucP (NanoLuc®-PEST) reporter gene 180–818SV40 late poly(A) signal 858–1079SV40 early enhancer/promoter 1127–1545Synthetic hygromycin (Hygr) coding region 1570–2607Synthetic poly(A) signal 2631–2679Reporter Vector primer 4 (RVprimer4) binding region 2746–2765ColE1-derived plasmid replication origin 3003Synthetic β-lactamase (Ampr) coding region 3794–4654Synthetic poly(A) signal/transcriptional pause site 4759–4912Reporter Vector primer 3 (RVprimer3) binding region 4861–4880

Sample Protocol to Determine Induction of Luciferase by TNFα inHEK 293 Cells Transfected with thepNL3.2.NF-κB-RE[NlucP/NF-κB-RE/Hygro] VectorMaterials to be Supplied by User• Dulbecco’s PBS (DPBS)• 0.05% (w/v) trypsin in DPBS• DMEM• DMEM supplemented with 10% fetal bovine serum (DMEM/FBS)• Tumor necrosis factor-α (Sigma T0157), 10µg/ml solution in PBScontaining 1mg/ml BSA• Nano-Glo® Luciferase Assay System (Cat.# N1110)• HEK 293 cells• Transfection reagent (e.g., FuGENE® HD Transfection Reagent, Cat.# E2311)Day 1: Plate Cells1. Grow HEK 293 cells in DMEM/FBS to approximately 75% confluency.2. Harvest cells via trypsinization: Remove the DMEM/FBS, wash the cells withDPBS and add the trypsin/DPBS (1X volume). After 2 minutes, add a 4X volumeof DMEM/FBS, collect the cell suspension and pellet the cells by centrifugation.Aspirate the supernatant and resuspend in DMEM/FBS. We have routinely used aconcentration of 15,000 viable cells/100µl DMEM/FBS.3. Dispense 100µl of the cell suspension into the wells of a 96-well plate. Plate enoughwells to perform each test condition in triplicate.4. Cover the plate and place it in a tissue culture incubator at 37°C overnight (or for24 hours).Day 2: Transfect Cells1. Transfect the cells using a high-efficiency transfection reagent (e.g., FuGENE® HDTransfection Reagent, Cat.# E2311). Transfection conditions may requireoptimization.2. Cover the plate and place it in a tissue culture incubator at 37°C overnight or asneeded for cell recovery depending on the transfection method used. We have used24 hours recovery time for lipid-mediated transfections.Day 3: Induce Transfected Cells1. Prepare 1X induction and 1X control solutions. Calculate the volume of 1X inductionand 1X control solution by multiplying the number of wells needed for each solutionby 100µl and prepare 110% of this amount.• 1X induction solution: Dilute 10µg/ml TNFα solution to 20ng/ml in DMEM/FBS.Final TNFα concentration will be 20ng/ml.• 1X control solution: DMEM/FBS.2. Remove media from wells that will be treated with either 1X induction solution or1X control solution.3. Add 100µl of 1X induction solution to the cells to be induced and 100µl of1X control solution to the control noninduced cells.4. Return the plate to the tissue culture incubator and induce for 5 hours.5. Analyze luciferase activity using the Nano-Glo® Luciferase Assay System(Cat.# N1110, Technical Manual #TM369).6. Calculate the fold induction as follows:Fold Induction = Average relative light units of induced cellsAverage relative light units of control cells

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