TAP1, TAP2, TAPBP and PSMB9 promoter sequences were amplified from genomic DNA and then cloned into the pGl3 luciferase (luc) vector (Promega, Fitchburg, USA) as recently described [20 (link), 48 (link)]. For transient transfections, 1 × 105 cells were cultured in 100 µl OptiMEM (Invitrogen), followed by transfection with 0.3 µg promoter constructs and 0.016 µg β-galactosidase (β-gal) vector using Lipofectamine 2000 (Invitrogen, Waltham, USA) as transfection reagent according to the manufacturer’s instructions. 48 h after transfection, the luc activity was determined by adding the luc substrate (Promega) using a luminometer and normalized to the transfection efficiency determined by ß-gal enzyme activity. Experiments were independently done three times using triplicates.
Pgl3 luciferase luc vector
Manufactured by Promega
Sourced in United States
The PGl3 luciferase (luc) vector is a plasmid containing the luciferase gene from the firefly Photinus pyralis. The luciferase gene is used as a reporter gene to study gene expression and regulation in various cell types and organisms.
Automatically generated - may contain errors
2 protocols using pgl3 luciferase luc vector
1
Quantifying Promoter Activity via Luciferase Assay
2
Promoter Activity Quantification
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TAP1/LMP2, TAP2 and TPN promoter sequences were amplified from genomic DNA and then cloned into the pGl3 luciferase (luc) vector (Promega, Fitchburg, USA) as recently described.46 (link) For transient transfections, 1 × 105 cells were incubated overnight in 100 µL OptiMEM (Invitrogen) followed by transfection with 0.3 µg of the respective promoter constructs and 0.016 µg of the β-galactosidase (β-gal) vector using Lipofectamine 2000 (Invitrogen, Waltham, USA) according to the manufacturer’s instructions. Forty-eight hours after transfection, the luc activity was determined with the luc substrate (Promega) using a luminometer and normalized to the transfection efficiency determined by β-galactosidase (ß-gal) enzyme activity.47 (link)
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