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...viability. Unlike intracellular labeling, dye staining can be done on live cells. For cellular proliferation... epitopes of interest that are found in the extracellular space. The targeted epitopes can be motifs within... glycosylation patterns. Surface labeling is particularly useful for classifying and sorting cells by ...technique that combines a marker with a signal. For clarity, Addgene uses 'labeling' to refer to antibodies...commonly used in western blotting or ELISAs.  A popular method for indirect labeling employs a primary ...further (non-lineage) markers alongside, e.g., for cellular activation. In addition, FACS achieves greater... T cell. Created using biorender.com.   Intracellular labeling Oftentimes, a marker of interest is...

...many postdocs this will lead to a raise in salary, particularly given that this has precipitated increases...institutional responses to the FLSA ruling. Similarly, from purely a salary perspective, while this is a move in...Many thanks to our guest blogger, David T. Riglar. David T Riglar is a postdoc at Harvard Medical School....post was contributed by guest blogger, David T. Riglar Advocating for Science Symposium and Workshop 2016...a joint American Society for Biochemistry and Molecular Biology/ FoR Hack Day during which several projects...continue their careers in science-related fields. In particular, two recent events have the potential to influence.... The topic of unionization continues to be a polarizing one. High-profile universities remain outspoken...

... the cellular level, it cannot be used for subcellular analysis such as morphology or subcellular localization...antibodies. It’s also particularly useful for intracellular staining where binding of large immune complexes...sources that allow users to assess a cell’s size, granularity, and target protein composition. It is used for...sheath fluid and transported through tubes or capillaries to a laser. As the cells move through the fluidics...Figure 1).   Figure 1: Cells expressing a particular surface receptor are stained with a fluorescent...information about the relative size of the cell; larger cells produce more FSC. Light that scatters sideways... different colors. This lends flow cytometry particularly well to complex experiments looking at a variety...

... as proxies for a cellular process or directly measure a cellular process. Cellular phenomena such as ...cell!)—ATP. There are biosensors for intracellular, extracellular, and even the ADP:ATP ratio. Most of... measure real-time cellular reactions to stimuli and as well as changes in cellular dynamics during so-called...biosensor is the thing that you care about within your cellular or animal system. This ‘thing’ could be a neurotransmitter... these ions, calcium is by far one of the most popular, with dozens of sensors developed for it. Calcium...These ion sensors can be targeted to different cellular locations or tissues depending on the biology ...of different factors, in different or the same cellular location, all at once! Small molecules What type...

...including monitoring multiple cellular events such as the dynamics of subcellular structures and gene expression...other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many intracellular sensors...luminescence. Intracellular Calcium Sensor Yang, J. et al. (7) wished to trigger intracellular Ca++ release...photoactivatable, optogenetic accuators to depolarize or hyperpolarize membranes. Traditional FRET reporters... sensor, membrane depolarization increases the BRET signal whereas hyperpolarization decreases the signal...orange fluorescent protein, CyOFP1. CyOFP1 was molecularly evolved from mNeptune2 through 33 mutations and...brightness and improved maturation time compared to similar orange fluorescent proteins. In their work, the...

... less selective if used in intracellular labeling because the intracellular compartment may contain cross-reacting...for the same clone. This can be particularly useful when designing larger multicolour panels, where different...approaches available broadly map onto the five pillars of antibody validation (see: Uhlen et al., 2016...for other sample types and for non-surface (intracellular) antigens. Unfortunately, antibodies frequently...protocol. This requires use of one of the five pillars of antibody validation, and it is recommended to...likely to express the target at a robust level, similar to that in samples of interest. If you are working...This can involve cell surface labeling and/or intracellular labeling (or both). It is important to test ...

...challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al., 2017) and ...be used to simultaneously target cellular RNA to specific intracellular locations using Cas9 and track ... satisfying ingredients reagents permeate the molecular biologist’s kitchen lab - and scientists using...molecule. Fluorescent RNA aptamers are used to track cellular processes by transforming cells with plasmids ...binding affinity to a specific target, and the molecular processes that make aptamer biology a powerful...a miniaturized version of Spinach that retains similar levels of fluorescence when bound to DFHBI despite...

...more efficiently amplified than larger constructs, but plasmids as large as ~6 kb can be amplified fairly...unmodified plasmid into a larger band (not shown). As a footnote; a similar approach can be used to identify... plasmid (or other). This is particularly relevant in cases where a large plasmid with multiple functional... to introduce a point mutation in a large construct. Particularly, if a pair of unique restriction site...the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data (see Xia...homologous ends recombine in vivo to give the final circular plasmid product pictured here. B) Screening of...portion of the plasmid containing the mutation). Similar results can be obtained with PCR screening using...

...for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University. No matter...for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University.     Additional...Primer on the Molecular Expressions Website Other major microscopy manufacturers have similar information...color contrast, to an image and thereby provide molecular or structural specificity to the viewer. Finally...thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished by...entire axial volume of a sample. Therefore, when a large number of optical slices are acquired all fluorophores...thin sheet of excitation light that propagates perpendicular to an imaging objective that collects fluorescence...

...combining them with a molecular species with high affinity for an endogenous molecular target, meaning the.... You may find yourself defaulting to the most popular or familiar options without even realizing it. ...attached to the target through binding of the molecular species. Administration of these dyes results ...fluorophores to enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent... dyes/ligands Effects on protein function and cellular components: It's important to assess how dye-conjugation...affects protein function and its propensity to bind cellular components. Some dyes target specific organelles.... Other dyes may prove challenging to stain intracellular compartments in in vivo model systems, so extra...