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Cytoplasm to Nucleus Translocation BioApplication |
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Catalog No. S50-0001-2 DescriptionSignal transduction is a complex process that involves activation and translocation of macromolecules, such as transcription factors from the cytoplasm to the nucleus, where the translocated macromolecules carry out their biological function, such as the transcription of target genes by transcription factors. Following signal transduction initiating events such as ligands binding to receptors, transcription factors such as NFkB, NFAT, C-jun, and STATs are typically activated and translocated to the nucleus. Thus a measure of the translocation of the transcription factor of interest from the cytoplasm to the nucleus is a measure of the activation of the transcription factor itself. In addition to transcription factors many kinases, upon phosphorylation, also undergo translocation in order to further transduce the signal along a pathway. The Cytoplasm to Nucleus Translocation BioApplication allows researchers to study any target of interest that translocates from the cytoplasm to the nucleus (or vice versa) and the user is only limited by the availability of a fluorescent label for the target of interest. For many such targets we provide validated HitKitTM HCS Reagent Kits, which can be used in conjunction with the Cytoplasm to Nucleus Translocation BioApplication. The Cytoplasm to Nucleus Translocation BioApplication measures translocation events by measuring the relative distribution of target fluorescence intensities between the cytoplasm region and the nuclear region of a cell. The BioApplication utilizes two-color fluorescence labeling of the cells. The BioApplication first identifies cells (objects) based on their fluorescent nuclear labeling (usually blue fluorescence). The BioApplication then defines a nuclear region and a cytoplasmic region of each cell, based on user defined assay parameter inputs. The BioApplication then measures fluorescent intensities in nuclear and cytoplasmic regions of each cell in the target channel (usually green fluorescence). The difference in fluorescent intensities between the nuclear and cytoplasmic regions of the cell is provided as an output feature (mean nucleus to cytoplasm intensity difference in target channel), which is an indicator of the magnitude of translocation of the target from the cytoplasm into the nucleus. The nucleus to cytoplasm intensity difference is low in cells which do not have any target signal in the nucleus (mostly cytoplasmic) and increases when the target translocates from the cytoplasm into the nucleus. The use of the Cytoplasm to Nucleus Translocation BioApplication to characterize the activation and nuclear translocation of the transcription factor NFkB in HeLa cells is shown below. HeLa cells plated in 96 well microplates were treated with varying concentration of interleukin-1 alpha (IL-1a) for up to 40 minutes. Cells were then fixed and stained using the reagents and protocol in the NFkB Activation HitKit HCS Reagent Kit. Figure 1 shows the translocation of NFkB from the cytoplasm (Panel B) to the nucleus (Panel C) following incubation of the cells with IL-1a. Figure 2 shows a dose response (left panel) plot of translocation of NFkB to the nucleus with different doses of IL-1a and the time course (right panel) of NFkB translocation following stimulation of HeLa cells with 1 ng/ml of IL-1a. The combination of HitKit HCS Reagent Kits and the Cytoplasm to Nucleus Translocation BioApplication provides a suite a validated assays for screening for kinase and transcription factor activity in the context of intact cells for higher quality information. 
Features- Characterizes translocation of a single biological target at the cell level
- Flexibility for user to choose any target of interest- limited only by availability of compatible antibody to target of interest
- Freedom to choose width of cytoplasmic area to be sampled
- Cell and well level output features include cytoplasm-to-nucleus intensity difference and also intensity in nuclear and cytoplasmic component regions
- BioApplication validated for screening
- Allows gating of sub-population of cells based on average and total intensities in target channel
Benefits- Provides information on whole, intact cells
- Streamlines transcription factor assays
- Analyzes subpopulations of cells for better biological information
Click the link below to view available product brochure
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