Fluorescent Antibody Probing
Fluorescent Antibody Probing - Related Videos
04:02
An Assay to Measure the Accumulation of a Fluorescent Antibiotic Probe in Bacterial Cells
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2025
This video demonstrates an assay to measure the accumulation of a fluorescent antibiotic probe in bacterial cells. Exposure to an efflux pump inhibitor disrupts the transmembrane proton gradient in bacterial cells, upon which a fluorophore-conjugated antibiotic is added. Inhibition of efflux pumps due to the disrupted proton gradient causes accumulation of the antibiotic inside the bacteria. Bacterial cells are lysed, and the fluorescence intensity is measured to confirm antibiotic accumulation.
10:59
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
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Cited by 9 •
2012
A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.
11:27
Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies
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Cited by 2 •
2008
We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.
06:48
Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads
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Cited by 3 •
2016
The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.
11:21
Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells
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Cited by 5 •
2010
This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.
FAQs
Direct probing uses a fluorophore-conjugated primary antibody that binds the target antigen itself. Indirect probing separates recognition from detection: an unlabeled primary antibody binds the antigen, and a fluorescent secondary antibody recognizes that primary antibody. This distinction gives researchers different labeling arrangements for visualizing the same type of molecular target in a biological sample.
The fluorophore must be illuminated at an excitation wavelength that stimulates fluorescence, after which it produces detectable emission. Fluorescence microscopy captures this emitted signal to reveal where antibody-bound targets occur. Thus, the optical setup connects the dye’s behavior with the observed image, allowing molecular labeling to be translated into spatial information within cells or tissues.
Signal location indicates the distribution of the recognized antigen within the examined sample. Researchers can therefore assess whether a protein, cellular structure, infection marker, or signaling-related target appears in particular cells or regions. The method provides spatial context rather than only indicating that the antigen is present, which is important for interpreting biological organization and changes.
Nuclear or organelle stains can be used alongside antibody-based fluorescence to provide structural reference points. Comparing the target signal with these labeled cellular features helps researchers relate antigen distribution to nuclei, organelles, or broader cell organization. This combined view strengthens interpretation by placing molecular localization within recognizable anatomical or cellular contexts.
A workflow begins with a biological sample containing the antigen of interest, followed by selection of either a fluorophore-conjugated primary antibody or an unlabeled primary antibody paired with a fluorescent secondary antibody. After antibody binding, fluorescence microscopy illuminates the sample and detects emitted signal, producing an image of target localization for analysis.
The technique is useful when researchers need both target specificity and spatial information. Applications described for it include cell biology studies of protein distribution and signaling-related changes, tissue or pathology investigations, detection of infection markers, and molecular diagnostics. These uses allow a labeled antigen to be examined within the organization of cells, tissues, or other biological samples.