Ratiometric Bimolecular Beacon
Ratiometric Bimolecular Beacon - Related Videos
12:20
Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
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Cited by 1 •
2014
Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.
04:29
Bimolecular Complementation Affinity Purification to Isolate Two Interacting Proteins
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2025
In this video, we have demonstrated bimolecular complementation affinity purification, BiCAP, to detect and isolate specific protein dimers using conformation-specific single-domain antibodies.
13:05
Ratiometric Imaging of Extracellular pH in Dental Biofilms
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Cited by 24 •
2016
A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.
15:01
Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
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Cited by 31 •
2012
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.
08:54
Bimolecular Fluorescence Complementation
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Cited by 66 •
2011
The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.
FAQs
Target binding changes the spatial relationship between the two labeled oligonucleotides. When each component hybridizes to an adjacent region of the same nucleic-acid target, the labels are brought close enough for fluorescence signal transfer, such as Förster resonance energy transfer. In the unbound condition, separation or a less favorable configuration limits that transfer, creating a target-dependent optical change.
The ratiometric readout is valuable because it uses one emission signal as a reference for another rather than relying on a single absolute intensity. This normalization can lessen the influence of probe concentration, illumination, and delivery differences. Consequently, the measured ratio can provide a more reliable indication of target-associated signaling when experimental conditions vary across samples or cellular locations.
Both oligonucleotide components contribute to sequence recognition, but their arrangement is especially important: each must find a neighboring target region so binding brings the labels into proximity. This requirement links the fluorescence change to the intended sequence architecture rather than merely to the presence of one short binding event. Interpreting the probe therefore requires considering adjacent target sites.
A basic measurement compares fluorescence from the two labeled components before and after exposure to a target sequence. The relevant change is not simply whether light is detected; it is the target-associated shift in the relationship between emission signals. Expressing that relationship as a ratio supports comparison across conditions affected by probe amount, illumination, or delivery.
Ratiometric Bimolecular Beacons can be applied to either RNA or DNA targets, depending on the sequence being investigated. In biology, this supports sequence-specific examination of gene expression and nucleic-acid localization. The approach is particularly useful when researchers need information about where a target occurs or how its presence varies in complex biological samples.
In complex samples, the ratio helps distinguish a target-related fluorescence change from variation caused by measurement conditions or probe delivery. Interpretation should therefore focus on the normalized signal associated with hybridization, while recognizing that the beacon reports the selected sequence rather than nucleic-acid abundance in general. This makes the method relevant to spatial and expression studies.