What is TR-FRET?

 

TR-FRET (Time Resolved Fluorescence Resonance Energy Transfer, Time-Resolved Fluorescence Resonance Energy Transfer) is a biophysical technique used to study intermolecular interactions. It combines two techniques: fluorescence resonance energy transfer (FRET) and Time-Resolved Fluorescence (TRF, Time-Resolved Fluorescence).

 

Schematic diagram of fluorescence resonance energy transfer

 

Fluorescence resonance energy transfer (FRET) is a physical phenomenon in which one fluorescent molecule (donor), after absorbing light energy, can nonradioactively transfer excitation energy to another molecule (acceptor) if the acceptor is spatially close enough to the donor (usually in the 1-10 nm range). The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, so it can be used to study the proximity and interactions between molecules.

 

Time-resolved fluorescence schematic diagram

 

Time-resolved fluorescence (TRF) is a technique for measuring fluorescence lifetime that distinguishes short-lived background fluorescence from long-lived signal fluorescence by measuring fluorescence decay after stopping the excitation light source. This allows fluorescence to be measured within a specific time window after excitation, thereby reducing background interference and improving the signal-to-noise ratio of the signal.

 

In TR-FRET, long-lived donor fluorophores, such as chelates of lanthanide elements (e.g. europium or terbium), are commonly used. These donors have a long fluorescence lifetime and can be detected within a delay time after excitation, when the short-lived background fluorescence has decayed. The acceptor fluorophore is typically an organic dye or another metal chelate that emits a different wavelength than that of the donor.

 

What can TR-FRET do?

 

Receptor binding studies:

For example, researchers may be interested in how a new drug binds to the beta-adrenergic receptor, a target of cardiac drugs. The β-adrenergic receptor may be labeled with Europium (Eu) as a donor and the drug molecule may be labeled with a suitable acceptor fluorophore.

The observed FRET signal increases when the drug binds to the receptor, allowing quantitative analysis of the binding affinity of the drug.

 

Enzyme activity test:

It was hypothesized to investigate the effect of a novel inhibitor on tyrosine kinase activity. The substrate of the kinase may be labeled with a donor fluorophore and the kinase may be labeled with an acceptor fluorophore. In the absence of an inhibitor, the kinase phosphorylates the substrate, causing the two to separate and the FRET signal to decrease. If the FRET signal remains high after the addition of the inhibitor, it is indicated that the inhibitor prevents kinase activity.

 

Protein-protein interactions:

To investigate the interaction between tumor suppressor protein p53 and MDM2. p53 was labeled with a donor fluorophore and MDM2 was labeled with an acceptor fluorophore. When p53 interacts with MDM2, FRET signaling increases and can be used to study the dynamics and regulatory mechanisms of this interaction.

 

Drug screening:

During drug development, TR-FRET can be used to high-throughput screen compound libraries to find potential drug candidates that bind to specific targets.

For example, a G protein coupled receptor (GPCR) can be screened for compounds capable of activating or inhibiting the receptor by labeling the receptor with donor and acceptor fluorophores.

 

What are the advantages and disadvantages of TR-FRET compared to ELISA?

 

abs7311 microplate (384-well plate, white, shallow well)

 

Compared to ELISA, TR-FRET has the following advantages:

★ High sensitivity: TR-FRET technology uses long-lived fluorescent labels (such as lanthanide elements) to detect signals for a longer time, thereby improving the sensitivity of detection;

★ High stability: Because of the use of time-resolved detection, background noise can be reduced and signal stability can be improved;

★ No washing steps required: TR-FRET experiments are usually homogeneous and require no washing steps, simplifying the operation flow;

★ Save samples: TR-FRET with 96-well plate (abs7308 microplate (96-well plate, white, shallow well) or 384-well plate (abs7311 microplate (384-well plate, white, shallow well), 384-well plate generally only requires 10uL sample per well;

★ Wide linear range: high-concentration and low-concentration samples can be detected simultaneously;

★ Fast: TR-FRET experiments can usually be completed within 1 h, including signal generation and detection.

 

However, since TR-FRET experiments require special fluorescent labeling and a time-resolved fluorescent plate reader (TRF Reader), this may be more expensive than traditional ELISA. At the technical level, TR-FRET technology may require more technical knowledge and professional equipment to conduct experiments and data analysis.

 

What are the TR-FRET products that absin can provide to its customers?

 

In addition to the 96-well and 384-well microplates mentioned above, absin can also provide assay kits for the Human KRAS & cRAF series, the Human IgG series, and the DDB1-CRBN & GSPT1 series.

 

1. Human KRAS & cRAF series

 

 

1) Labor-saving: no washing required;

2) Time-saving: only 1 hour of incubation is required;

3) Provincial sample: 20uL/well or lower reaction system;

4) High throughput: 96/384/1536 microplates can be matched.

 

Item number	Product Name	Specifications
abs560003	Human KRAS WT & cRAF Binding Kit	500T
abs560004	Human KRAS G12C & cRAF Binding Kit	500T
abs560005	Human KRAS G12D & cRAF Binding Kit	500T
abs560006	Human KRAS G12R & cRAF Binding Kit	500T
abs560007	Human KRAS G12V & cRAF Binding Kit	500T
abs560008	Human KRAS G13C & cRAF Binding Kit	500T
abs560009	Human KRAS G13D & cRAF Binding Kit	500T
abs560010	Human KRAS Q61H & cRAF Binding Kit	500T

 

Data example (abs560004 Human KRAS G12C & cRAF Binding Kit as an example):

The following data is not a substitute for the data obtained in the experiment and is only an example and the results may vary depending on the TR-FRET compatible instrument.

 

 

2. Human IgG Series

 

abs560002 Human Total IgG Kit (Assay Pro) Competition Law

 

abs560011 Human IgG Sandwich Assay Kit Sandwich method

 

Item number	Product Name	Specifications	Sample concentration range
abs560002	Human Total IgG Kit (Assay Pro)	100T/500T/10000T	1.22-80000ng/mL
abs560011	Human IgG Sandwich Assay Kit	100T/500T/5000T/10000T	3.9-4000ng/mL

 

Data instance

The following data is not a substitute for the data obtained in the experiment and is only an example and the results may vary depending on the TR-FRET compatible instrument.

 

✧ abs560002 Human Total IgG Kit (Assay Pro) Competition Law

 

 

✧ abs560011 Human IgG Sandwich Assay Kit Sandwich method

 

 

3. Human DDB1-CRBN & GSPT1 series

 

 

It is used to determine the interaction between the DDB1/CRBN complex and GSPT1 mediated by the molecular gel CC-885 and the compound to be detected. It is a high-throughput method to achieve simple and rapid detection of small molecules capable of mediating the interaction between the DDB1/CRBN complex and GSPT1.

 

Item number	Product Name	Specifications
abs560012	Human DDB1-CRBN & GSPT1 Binding Kit	500T

 

Data Example

The following data is not a substitute for the data obtained in the experiment and is only an example and the results may vary depending on the TR-FRET compatible instrument.

 

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.net

Follow us on Facebook: Absin Bio