Looking to improve the performance of your protein assays? Our high-quality single quenched fluorescent peptides and FRET peptide libraries can provide you with the insights you need. Whether you are studying enzyme specificity or using FRET peptides as reagents for biosensing or subcellular imaging, we have the solutions that fit your unique needs. Our advanced peptide conjugation and modification capabilities allow us to source the best fluorophores and quenchers and quickly conjugate them and deliver them to your lab without sacrificing quality.
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Quenched fluorescent peptides, also known as “FRET peptides”, are commonly used to measure enzyme activity, protein-protein interactions, and subcellular protein trafficking, among others. FRET stands for Förster or Fluorescence Resonance Energy Transfer, a method designed to detect distance-dependent interactions between different molecules linked to dyes.
Peptides used in these studies are created by conjugating synthetic protein segments with both a fluorophore and quencher dye with overlapping spectra. Due to the overlap, when the two dyes are in close proximity (within 1-10 nm), the quencher can effectively block the light emitted by its fluorescent counterpart.
The fluorophore is activated only when the distance between the two dyes increases, which is often caused by enzymatic degradation, allowing the detection and quantification of activity. This property makes quenched fluorescent peptides ideal substrates for real-time monitoring of protein activity, providing detailed insights into mechanisms of action.
Are you looking to expedite your protein research? Our team of experts can provide you with personalized solutions to help streamline your projects and achieve your research goals. Whether you need single FRET peptides or are interested in designing and synthesizing entire FRET peptide libraries, our team is here to help.
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Quenched fluorescent peptides are commonly used in:
Besides enzyme studies, FRET peptides are invaluable reagents for subcellular imaging, both in vitro and in vivo. When tracking subcellular activity and reactions, quenched fluorescent peptides are typically attached to a specific biomolecule or organelle. These experiments provide in-depth insights into the dynamics of cellular processes, such as signaling pathways or protein trafficking.
In the context of disease, FRET peptides have been employed in vivo to provide insights into specific disease mechanisms. For instance, these reagents have been frequently used to unveil the process of synthesis of amyloid-beta, which has been associated with a marked decline in cognitive function in Alzheimer’s disease.
In the context of drug development, quenched fluorescent peptides provide the opportunity to both discover new drug targets or test the pharmacodynamics of new candidate drugs.
Quenched fluorescent peptides have also garnered interest as reagents for biosensing. The advantage of FRET reagents over conventional biochemical assays is simple. The former allows the optical measure of activity, enabling the investigation of live cells in a non-destructive and, often, minimally invasive way.
Unlike other applications, FRET biosensing assays can employ different strategies, either the fluorophore and quencher are attached to the same molecule (intramolecular biosensing), or the two dyes are fused to different molecules (intermolecular biosensing). In intermolecular assays, FRET changes occur when the two independent molecules come into close proximity. In this case, activity can be measured as a loss instead of a gain in brightness.
FRET peptides are simple and inexpensive to synthesize, and they can be used in a variety of different setups at different scales, ranging from immobilized to solution-based, making them versatile tools for enzyme research, drug development, biosensing, and fundamental functional studies.
Many different fluorophore-quencher pairs can be used in the synthesis of FRET peptides. The choice of the best pair depends on factors such as the intended application, availability and cost of the dyes, and desired sensitivity and resolution of measurements.
For instance, in vivo applications require careful consideration of dye toxicity as the component may modify the cellular process researchers are attempting to measure, while cell-free experiments can make use of slightly cytotoxic FRET pairs if a higher sensitivity is desired.
The most commonly used FRET molecules pair variants of cyan fluorescent protein (CFP) with yellow fluorescent protein (YFP). These two families of dyes are readily available, easy to obtain, compatible with a variety of experimental techniques, and accessible to researchers who are aiming at getting more insights at a lower cost for their assays.
Given the success and availability of CFP dyes, today they represent a large family of reagents, which includes mTurquoise2, mCerulean3, mTFP1, and Aquamarine. These engineered cyan donors have long fluorescence lifetimes, thus, making them advantageous for a wide range of experimental settings.
Are you looking for a more sensitive and accurate way to investigate the substrate specificity of proteases? Internally quenched fluorescent (IQF) peptides may be the answer. These specialized peptides are derived from FRET peptides and offer improved analytical performance by using non-fluorescent quenchers. IQF peptides can also contain unnatural amino acids, allowing them to be used to investigate protease specificity in peptides with post-translational modifications.
The most commonly used IQF/FRET substrate pairs include:
Although Dabcyl has remained a widely used quencher, studies have shown that its hydrophobic nature may hamper solubility. Its high cost and bulky structure, prone to interacting with protease binding pockets, makes it less suitable for complex and vast screening experiments.
In contrast, DNP is inexpensive and smaller than Dabcyl, making it easier to incorporate into peptide substrates and easier to solubilize. Thus, in recent years, DNP-based quenchers are gaining ground as the preferred non-fluorescent quenchers in IQF peptides.
Make the switch to IQF peptides and improve the performance of your protease activity and specificity experiments today:
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