The LightSwitch™ Luciferase Assay Kit was designed to provide superior results and be easy to use. It utilizes our novel, proprietary LightSwitch Assay Substrate, which was formulated specifically for use with our engineered RenSP luciferase gene. In addition, the kit's Assay Buffer makes possible one-step reagent addition, eliminating the need to perform a separate lysis step. The LightSwitch Assay offers a number of advantages:
- Quantitative – Achieve high sensitivity and a broad dynamic range
(see the Data tab below) - Convenient – Simply mix reagents, add directly to cells, then read on a luminometer
- Optimized – Get optimal results with LightSwitch assay reagents, developed specifically for use with our LightSwitch Reporter constructs
- Flexibility – Use for a small number of samples, or easily scale up to perform thousands of assays
- Products (1)
- Documents (12)
- LightSwitch™ Luciferase Assay Overview
- 3´UTR Controls
- Promoter Controls
- pLightSwitch Vectors
- Kit Contents
- Data
Name | Format | Cat No. | Price | |
---|---|---|---|---|
LightSwitch™ Luciferase Assay Kit | 100 assays | 32031 | $185 | Add to Cart |
1000 assays | 32032 | $875 | Add to Cart |
The LightSwitch Luciferase Assay System is a unique collection of over 30,000 regulatory elements that are available in the LightSwitch Promoter and 3´UTR Collections. After selecting a pre-cloned LightSwitch Promoter or 3´UTR Luciferase Reporter Vector, it is transfected into an appropriate cell line. The cells are stimulated, if required, to induce transcription of RenSP luciferase. Gene expression is then quantified using a LightSwitch Assay Kit and a luminometer to measure the amount of luminescence produced.
Advantages of the LightSwitch Luciferase Assay System
- Quantitative – Novel RenSP luciferase technology allows you to measure promoter activity with industry-leading sensitivity and dynamic range.
- Simple, fast, complete solution – With pre-cloned LightSwitch Reporter vectors and optimized transfection & assay reagents, you can study regulation of your gene today. No cloning, DNA preparation or optimization is needed, and most studies do not require any internal transfection controls.
- Comprehensive and verified – The genome-wide LightSwitch Reporter Collections are sequence-verified, transfection-ready promoter and 3´UTR reporter vectors.
- Cost-effective – Efficiently screen for activation and/or repression using a multitude of conditions.
RenSP – maximum brightness and minimal background
Why use Renilla luciferase?
Marine luciferases have become popular alternatives to firefly luciferase as a genetic reporter based on assay simplicity, high sensitivity, and a broad linear range of signal that provides greater sensitivity over firefly luciferases.1,2 The Renilla luciferase protein catalyzes oxidation of its coelenterazine substrate in the reaction shown below to produce light at 480 nm, easily read by standard luminometers.3
Inherent advantage of Renilla luciferase
Because marine bioluminescence has evolved independently many times, a variety of luciferases target the same substrate (coelenterazine) yet bear little resemblance to one another.4 Renilla reniformis is a sea pansy that responds to mechanical stimulation by generating a blue-green bioluminescence.4 The small size of its gene and protein (936 bp and 36 kD) and its lack of dependence on ATP provide Renilla luciferase with a distinct advantage over larger, ATP-dependent luciferases like those from fireflies (~1.6 kb and 62 kD).5-8
Figure 1: Light is produced from coelenterazine by Renilla luciferase.
Optimizing Renilla for LightSwitch reporter assays – increased enzymatic activity and brightness
We have created an optimized Renilla luminescent reporter gene, called RenSP, by increasing its overall enzymatic activity (light output) and adding a protein destabilization domain to decrease the half-life of the RenSP protein. Starting with a base sequence of the native Renilla gene, we functionally screened thousands of synthetic gene sequence variants that included a variety of predicted improvements. We also removed transcription factor binding sites from the gene sequence as these might confound expression measurements. As a result, we created the RenSP luciferase that is 100% brighter than other humanized versions of Renilla luciferase (Figure 2).9
Figure 2: The absolute signal of RenSP is significantly brighter than hRlucP.
Enhanced degradation rate of RenSP improves assay response
One limitation of reporter gene assays is that the reporter protein can accumulate in the cell; this can delay and dilute the measurable response to stimulation or repression. To eliminate this problem, the RenSP gene has been fused to a protein destabilization domain to reduce the accumulation of reporter protein. The RenSP reporter gene contains a PEST protein degradation sequence from mouse Ornithine Decarboxylase (mODC) that has been shown to increase rates of protein turnover.10-13
The destabilized RenSP luciferase protein has a half-life of approximately 1 hour compared to the ~3 hour half-life of the native luciferase protein, the CAT reporter protein half-life of ~50 hours and the GFP half-life of 25 hours. The RenSP-PEST fusion protein therefore combines the benefits of increased signal with a short half-life reporter to provide a sensitive measure of the induction or repression of reporter gene activity. Figure 3 highlights an example in which signal knock-down of a UTR reporter after addition of a miRNA is more easily detected when the target UTR is fused to a PEST-containing reporter gene.
Figure 3: RenSP with PEST increases the knock-down of 3´UTR targets in the presence of mir-122.
Empty 3´UTR vector: The empty 3´UTR vector contains the RenSP luciferase reporter gene and its constitutive promoter. This construct may serve as a positive control for the transfection because of its promoter. It may also be utilized for creating additional constructs. For a vector map, annotations and primer & restriction sites, please see the Empty LightSwitch™ Reporter Vectors page.
Housekeeping gene 3´UTR vectors: Housekeeping control constructs contain the 3´UTRs for common housekeeping genes cloned downstream of the RenSP luciferase reporter gene.
Random control 3´UTR vectors: Random control constructs contain non-conserved, non-genic, and non-repetitive human genomic fragments.
3´UTRs of housekeeping genes
Catalog No. | Name | Description | Coordinates | Sequence |
---|---|---|---|---|
32013 | LightSwitch™ ACTB 3´UTR Control |
Beta-actin | chr7–: 5340002 - 5340681 |
Get Info |
32014 | LightSwitch™ GAPDH 3´UTR Control |
Glyceraldehyde-3-phosphate dehydrogenase |
chr12+: 6517534 - 6517833 |
Get Info |
32015 | LightSwitch™ LDHA 3´UTR Control |
Lactose dehydrogenase A | chr11+: 18385382 - 18386064 |
Get Info |
32016 | LightSwitch™ PPIA 3´UTR Control |
Peptidylprolyl isomerase A | chr7+: 44614226 - 44616012 |
Get Info |
Random genomic fragments in 3´UTR vector
Catalog No. | Name | Coordinates | Sequence |
---|---|---|---|
32017 | LightSwitch™ Random 3´UTR Control 1 |
chr13+: 82190847 - 82193385 |
Get Info |
32018 | LightSwitch™ Random 3´UTR Control 2 |
chr12+: 82120892 - 82121673 |
Get Info |
32019 | LightSwitch™ Random 3´UTR Control 3 |
chr13+: 26144830 - 26145608 |
Get Info |
32020 | LightSwitch™ Random 3´UTR Control 4 |
chr22+: 1645930 - 1646850 |
Get Info |
Empty promoter vector: The empty promoter vector contains the RenSP luciferase reporter gene without a promoter. This construct serves as a measure of background signal in the experiment. This construct may also be used to create additional experimental constructs. For a vector map, annotations and primer & restriction sites, please see the Empty LightSwitch™ Reporter Vectors page.
Housekeeping gene promoter vectors: Housekeeping control constructs contain promoters for common housekeeping genes driving the RenSP luciferase reporter gene. These constructs serve as positive transfection controls and may also serve as controls for comparing signals between conditions if they are known to be unresponsive to the test condition.
Random control promoter vectors: Random control constructs contain 1 kb non-conserved, non-genic, non-repetitive fragments from the human genome cloned upstream of the RenSP luciferase reporter gene. These vectors produce slightly different signals than the empty promoter vector and are considered optimal negative or background controls.
Promoters of housekeeping genes (Positive controls)
Catalog No. | Name | Description | Coordinates | Sequence |
---|---|---|---|---|
32003 | LightSwitch™ ACTB Promoter Control |
Beta-actin | chr7–: 5343428 - 5344428 |
Get Info |
32004 | LightSwitch™ GAPDH Promoter Control |
Glyceraldehyde-3-phosphate dehydrogenase |
chr12+: 6513163 - 6514226 |
Get Info |
32005 | LightSwitch™ LDHA Promoter Control |
Lactose dehydrogenase A | chr11+: 18372059 - 18372891 |
Get Info |
32006 | LightSwitch™ RPL10 Promoter Control |
Ribosomal protein L10 | chrX+: 153146645 - 153147658 |
Get Info |
Random genomic fragments in promoter vector (Negative controls)
Catalog No. | Name | Coordinates | Sequence |
---|---|---|---|
32007 | LightSwitch™ Random Promoter Control 1 |
chr11+: 119015626 - 119016592 |
Get Info |
32008 | LightSwitch™ Random Promoter Control 2 |
chr1+: 226079717 - 226080649 |
Get Info |
32009 | LightSwitch™ Random Promoter Control 3 |
chr6+: 15604416 - 15605350 |
Get Info |
32010 | LightSwitch™ Random Promoter Control 4 |
chr16+: 1645930 - 1646850 |
Get Info |
LightSwitch Promoter Reporter Vector
Our scientists created the LightSwitch Promoter Reporter Collection by using an algorithm to evaluate over 5 million human cDNA sequences in order to identify transcription start sites (TSS) throughout the human genome. Based on these TSS predictions, over 18,000 human promoter sequences of ~1 kb (-900…+100 bp relative to the TSS) were cloned into the pLightSwitch_Prom reporter vector. If you cannot find the promoter you wish to study when you search the LightSwitch Promoter Reporter Collection, you can clone DNA fragments that contain promoter sequences into the MCS of pLightSwitch_Prom in order to measure promoter activity.
View the vector sequence and annotation file (txt) for the pLightSwitch_Prom vector.
Promoter Insert Sequencing Primers:
Forward: TCCATCAAAACAAAACGAAACAA
Reverse: AGTCGAGCACGTTCATCTGCTT
LightSwitch 3´UTR Reporter Vector
Following systematic identification of 3´UTR sequences in the human genome using RefSeq and other cDNA resources, we created the LightSwitch 3´UTR Reporter Collection by cloning over 12,000 human 3´UTRs ranging in size from 300 bp to 3,000 bp into the pLightSwitch_3UTR vector. If you cannot find the 3´UTRs you wish to study when you search the LightSwitch 3´UTR Reporter Collection, you can clone 3´UTR sequences into the MCS of pLightSwitch_3UTR. As cloned fragments are downstream of the RenSP luciferase reporter gene, they become part of a hybrid transcript that contains the luciferase coding sequence fused to the UTR sequence of interest.
View the vector sequence and annotation file (txt) for the pLightSwitch_3UTR vector.
3´UTR Insert Sequencing Primers:
Forward: GGGAAGTACATCAAGAGCTTCGT
Reverse: CCCCCTGAACCTGAAACATAAA
LightSwitch Long-range Enhancer Reporter Vector
If one of the optimized synthetic regulatory elements found in the LightSwitch Synthetic Response Elements collection is not appropriate for your research, DNA fragments that contain long-range enhancer elements can be cloned into the MCS of the pLightSwitch_LR vector, which is immediately upstream of a basal TK promoter that transcribes the RenSP luciferase reporter gene. Changes in luciferase levels can then be measured to determine the impact of the cloned enhancer element on transcriptional activity.
View the vector sequence and annotation file (txt) for the pLightSwitch_LR vector.
LR Insert Sequencing Primers:
Forward: TCCATCAAAACAAAACGAAACAA
Reverse: AGTCGAGCACGTTCATCTGCTT
LightSwitch 5´UTR Reporter Vector
You can clone DNA fragments that contain 5´UTR sequences into the MCS of the pLightSwitch_5UTR vector. Fragments cloned into the MCS upstream of the RenSP luciferase reporter gene will become part of a hybrid transcript that contains the UTR sequence of interest fused to the luciferase coding sequence.
View the vector sequence and annotation file (txt) for the pLightSwitch_5UTR vector.
5´UTR Insert Sequencing Primers:
Reverse: AGTCGAGCACGTTCATCTGCTT
Contents & Storage
The LightSwitch™ Luciferase Assay Kit contains:
- Lyophilized 100X LightSwitch™ Assay Substrate (-20°C)
- LightSwitch™ Substrate Solvent (room temperature)
- LightSwitch™ Assay Buffer (-20°C)
- Protocol
All reagents are guaranteed stable for 6 months when stored properly.