Epitope tagging is a recombinant DNA method by which a protein encoded by a cloned gene is made immunoreactive to a known antibody. This review discusses the major advantages and limitations of epitope tagging and describes a number of recent applications. Major areas of application include monitoring protein expression, localizing proteins at the cellular and subcellular levels, and protein purification, as well as the analysis of protein topology, dynamics and interactions. Recently the method has also found use in transgenic and gene therapy studies and in the emerging fields of functional genomics and proteomics.
Echinobase (www.echinobase.org) is a third generation web resource supporting genomic research on echinoderms. The new version was built by cloning the mature Xenopus model organism knowledgebase, Xenbase, refactoring data ingestion pipelines and modifying the user interface to adapt to multispecies echinoderm content. This approach leveraged over 15 years of previous database and web application development to generate a new fully featured informatics resource in a single year. In addition to the software stack, Echinobase uses the private cloud and physical hosts that support Xenbase. Echinobase currently supports six echinoderm species, focused on those used for genomics, developmental biology and gene regulatory network analyses. Over 38 000 gene pages, 18 000 publications, new improved genome assemblies, JBrowse genome browser and BLAST + services are available and supported by the development of a new echinoderm anatomical ontology, uniformly applied formal gene nomenclature, and consistent orthology predictions. A novel feature of Echinobase is integrating support for multiple, disparate species. New genomes from the diverse echinoderm phylum will be added and supported as data becomes available. The common code development design of the integrated knowledgebases ensures parallel improvements as each resource evolves. This approach is widely applicable for developing new model organism informatics resources.
Live cell imaging
requires bright photostable dyes that can target
intracellular organelles and proteins with high specificity in a no-wash
protocol. Organic dyes possess the desired photochemical properties
and can be covalently linked to various protein tags. The currently
available fluorogenic dyes are in the green/yellow range where there
is high cellular autofluorescence and the near-infrared (NIR) dyes
need to be washed out. Protein-mediated activation of far-red fluorogenic
dyes has the potential to address these challenges because the cell-permeant
dye is small and nonfluorescent until bound to its activating protein,
and this binding is rapid. In this study, three single chain variable
fragment (scFv)-derived fluorogen activating proteins (FAPs), which
activate far-red emitting fluorogens, were evaluated for targeting,
brightness, and photostability in the cytosol, nucleus, mitochondria,
peroxisomes, and endoplasmic reticulum with a cell-permeant malachite
green analog in cultured mammalian cells. Efficient labeling was achieved
within 20–30 min for each protein upon the addition of nM concentrations
of dye, producing a signal that colocalized significantly with a linked
mCerulean3 (mCer3) fluorescent protein and organelle specific dyes
but showed divergent photostability and brightness properties dependent
on the FAP. These FAPs and the ester of malachite green dye (MGe)
can be used as specific, rapid, and wash-free labels for intracellular
sites in live cells with far-red excitation and emission properties,
useful in a variety of multicolor experiments.
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