Rfp Quantification
Rfp Quantification - Related Videos
06:07
A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila
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Cited by 1 •
2009
We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.
08:44
Invertebrate Lifespan Quantification
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2023
Many animals naturally stop growing upon reaching adulthood, after which they undergo aging or "senescence" until dying. The amount of time between an organism\'s birth and death is called its lifespan, which can be influenced by various biological and environmental factors. By exposing organisms to different growth conditions, scientists can better understand the factors affecting lifespan. Flies and worms are ideal organisms to perform such experiments, given their short generation time and...
16:59
Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture
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Cited by 14 •
2011
Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.
08:38
Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS
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Cited by 28 •
2015
Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.
11:54
Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator
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Cited by 9 •
2017
Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.
FAQs
The RFP chromophore must be excited at a defined wavelength so its emitted red fluorescence can be measured consistently. Background correction then removes signal that does not arise from the fluorescent protein itself. Without this correction, nonspecific fluorescence can inflate the apparent signal and weaken comparisons among biochemical samples or experimental conditions.
Calibrated standards or reference samples provide a basis for interpreting measured fluorescence rather than treating raw intensity as an absolute result. Comparing sample signals with these controls helps relate fluorescence to protein abundance, gene expression, or biological activity. This approach also supports more meaningful comparisons across biochemical conditions and experimental outcomes.
The sample format determines how fluorescence is collected and interpreted. Purified proteins provide a defined biochemical sample, whereas cell lysates represent fluorescent material released from cells. Living cells allow measurements in an intact biological setting and can support localization analyses. These formats therefore address different experimental questions even when the same red fluorescent signal is measured.
A typical workflow selects the sample type, excites the RFP chromophore at a defined wavelength, and records the emitted red fluorescence with an appropriate instrument. Researchers then correct the measurement for background and compare the result with calibrated standards or reference samples. The processed signal can be used to evaluate protein abundance, gene expression, or biological activity.
Plate readers, fluorometers, and imaging systems can all acquire RFP fluorescence, but they support different measurement formats. Plate readers and fluorometers are suited to fluorescence measurements from samples such as purified proteins or lysates, while imaging systems can measure fluorescence in living cells and support cellular localization analyses. Instrument selection should match the sample and intended outcome.
In reporter-gene assays, the measured red fluorescence provides a quantitative readout associated with gene expression or biological activity. In protein-expression studies, it helps estimate the abundance of an RFP-containing protein in biochemical samples. Comparing corrected signals with standards or reference samples allows researchers to evaluate differences among experimental conditions rather than relying only on visual fluorescence.
When fluorescence is measured in living cells with an imaging system, the red signal can be examined in relation to where fluorescent material appears within the cells. This extends the analysis beyond overall signal intensity by supporting cellular localization studies. Quantitative comparisons can help evaluate how localization-associated fluorescence changes across biochemical or experimental conditions.