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Fluorescent Proteins & Reporters


Products >  Fluorescent_Proteins_and_Reporters >  Fluorescent_Proteins >  Promoter_Reporters >  Promoterless_Lowest_Background

Fluorescent Protein Promoter Reporters—On Demand Reporting

On-demand reporter vectors are the next generation of promoter reporters. With these reporters systems, you can compensate effectively for reporter background without compromising your assay’s signal intensity. Low background and a bright signal are no longer mutually exclusive.

The Challenges: Overcoming High Background & Low Signal Intensity

Traditional promoter reporter assays generally struggle with the fact that most promoters are not very “tight.” As a result, your promoter of interest may drive reporter expression even without being activated—for example, during the time between transfection and the start of your experiment. These preexisting reporter molecules (the background) are the main cause of a low signal-to-noise ratio after promoter induction during the actual experiment.

A previous approach to this problem was to modify reporters for very quick, constitutive degradation. However, because these reporters are constitutively degraded as soon as they are made, it is impossible to accumulate a large quantity of reporter molecules inside the cell even upon promoter activation. As a result, only a fraction of the reporter molecules are present long enough to be measured, and this type of assay has low signal intensity.

The Solution: Reporters On-Demand

The On-Demand Living Colors Fluorescent Protein Reporter Systems meet the challenge by providing both a low background and a broad dynamic range. This versatility is possible because they use a combination of technologies: each system includes a bright fluorescent protein reporter (AmCyan1, tdTomato, or ZsGreen1) for high signal intensity, coupled with ligand-dependent ProteoTuner protein stabilization/destabilization technology to eliminate background.

In these systems, the fluorescent protein reporter is expressed as a fusion protein tagged on its N-terminus with a ligand-dependent destabilization domain (DD). The DD rapidly targets the reporter protein for proteasomal degradation, guaranteeing a low reporter background signal at the start of your experiment. However, when the small, membrane-permeant ligand Shield1 is added to the sample, it binds to the DD and protects the reporter from degradation, so that it can accumulate.

By adding Shield1 simultaneously with your candidate inducer, you can effectively stabilize the reporter protein when it is synthesized in response to promoter activation. The majority of the fluorescent protein reporter molecules expressed during promoter activation will contribute to your readout, allowing for a considerably higher dynamic range and drastically improved signal-to-noise ratio compared to other types of reporter systems.


On-Demand Fluorescent Protein Reporter Systems Traditional Reporter Systems
BackgroundUniformly low Promoter-dependent; may be high
SignalBrightReporter-dependent. If background is low, signal is usually dim
Signal-to-noise ratio High; due to bright signal and low background Often low, especially when background is low
Studying timely promoter activity by eliminating unwanted reporter molecules Easy—simply remove Shield1 reagent Difficult; depends on reporter's natural lifespan

High Signal, Low Background

In order to demonstrate the high signal-to-noise ratio and wide dynamic range of the DD-Fluorescent Protein Reporter Systems, we compared the fold induction achieved using the DD-fluorescent protein reporters with that achieved using regular (non-destabilized) fluorescent proteins. The DD-tagged reporters stabilized by Shield1 had a much wider dynamic range, and therefore a much larger fold increase in the signal than the untagged versions of the same reporters. For the untagged versions, we observed high background fluorescence from reporter molecules that accumulated prior to induction, which drastically reduced the fold increase in signal intensity.

Choose Your On-Demand Reporter System

Our On-Demand Reporter Systems each consist of the necessary vectors (red, green, or cyan) plus Shield1.

Flexible Choices

Use the DD Fluorescent Protein Reporter Systems to monitor any promoter of interest—just insert your promoter of interest upstream of the DD reporter. Choose from plasmid or lentiviral vector formats.

At-A-Glance Documents Images & Data Resources

Features

The next generation of promoter reporters

  • High signal-to-noise ratio
  • Bright signal
  • Easy to monitor—red, green, and cyan options

Additional Information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.


 
Products
Cat. # Product Package Size Price License # of Units Select
631089 CRE DD Cyan Reporter System Each $569.00 License Statements *
631085 CRE DD Green Reporter System Each $569.00 License Statements *
631087 CRE DD Red Reporter System Each $569.00 License Statements *
632191 DD-AmCyan1 Reporter System Each $569.00 License Statements *
632192 DD-ZsGreen1 Reporter System Each $569.00 License Statements *
632190 DD-tdTomato Reporter System Each $569.00 License Statements *
631748 Lenti-X™ DD Cyan Reporter System Each $1,096.00 License Statements *
631751 Lenti-X™ DD Green Reporter System Each $1,096.00 License Statements *
631753 Lenti-X™ DD Red Reporter System Each $1,096.00 License Statements *
631083 NFkappaB DD Cyan Reporter System Each $569.00 License Statements *
631079 NFkappaB DD Green Reporter System Each $569.00 License Statements *
631081 NFkappaB DD Red Reporter System Each $569.00 License Statements *
 
* You must log in if you would like to purchase these products online, since the purchase of these products may be restricted depending on your account type. Researchers at not-for-profit accounts receive a limited use license with their purchase of the product. Researchers at for-profit accounts must obtain a license prior to purchase. For details please contact licensing@clontech.com.

 



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