...Fluorescent protein fusions One of the easiest ways to track a given protein is to fuse it to a fluorescent protein...fluorescent signal and enables single molecule tracking  within living cells without affecting protein...thereby creating a single-molecule reporter of intracellular processes. Initially, Tanenbaum et al. observed...molecules very effectively - they even managed to track run lengths of the motor protein kinesin across ...cell physiology. Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function,...photobleaching Simplified, long-term single-molecule tracking Caveats of SunTag Very large size: a 24x scaffold...

... in the extracellular domain which activates the native signaling cascade of the intracellular part of...ligands (PORTLs). OptoXRs are chimeras of the extracellular domain of endogenous light-sensitive receptors...receptors, like mammalian rhodopsin, and the intracellular domain of an endogenous GPCR, such as adrenergic...signaling pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be...state.      Figure 4: Example of an intracellular signaling pathway activated by an optogenetic...., & Cui, B. (2015). Optogenetic control of intracellular signaling pathways. Trends in Biotechnology,...

...developed by Addgene depositors have made it easier to track translation in two different ways: by monitoring...monitoring the first round of translation or by tracking the translation of a single mRNA over time. Both are ...translation. Single molecule continuous translation tracking TRICK allows you to see the first round of translation...translation, but it doesn’t allow continued tracking of a given mRNA because NLS-PCP-GFP is removed by ...first ribosome that translates the mRNA. To permit tracking over time, Yan et al. devised a system that labels...keeps it in a single field of view for constant tracking. You can then directly monitor multiple rounds...

... endocytosis, vesicle trafficking, or muscle contractions) are produced by orchestrated movements of networks...machines R bodies can repeatedly extend and contract in response to changing pH, and they can rupture...force-generating protein machines that can extend and retract over many microns in response to pH changes without...penetrate other barriers (like vasculature or extracellular matrices) in response to pH changes. Beyond... PMCID: PMC213417. 2. Schrallhammer, M. et al. Tracing the role of R-bodies in the killer trait: Absence...

...antibodies. It’s also particularly useful for intracellular staining where binding of large immune complexes...antibody thus amplifying the signal.       Intracellular protein targets, measured by fixing and permeabilizing...permeabilizing membranes For intracellular protein targets, users must first fix proteins within the cells...fixation. If staining both cell surface and intracellular proteins in parallel, then stain the surface...first, fix and permeabilize, and then stain the intracellular target. When using this approach, avoid fixation-sensitive...inhibit secretion and then follow the typical intracellular staining protocol. Sorting cells with FACS In...fixation and permeabilization are required, as in intracellular staining, consider using an amine reactive dye...

...preparation, VSV-G is stable and can withstand ultracentrifugation which helps scientists achieve a high titer... axon terminals and can’t be used for neuronal tracing or peripheral gene delivery. Combining the rabies... rabies glycoprotein now serves as a retrograde tracer so that scientists can identify the neurons that...2001).  This method can also allow scientists to trace a neuron’s projections back to the cell body. Rabies...Conzelmann K-K, Callaway EM (2006) Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods...2007) Monosynaptic Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons. Neuron...

...reporters that use fluorophores and luciferases to track bacteria in real time, but each of these reporters...located deep in the body, like the gastrointestinal tract. Require Administration of Substrate: Some luciferase...signals in ultrasound imaging. Ultrasound is an attractive imaging platform for in vivo imaging experiments...ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain Nissle 197 (ECN) was used for...strain that can colonize the mammalian intestinal tract and has been used for 100 years to clinically treat...

...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...minutes in living cells, meaning that scientists can track the movement and localization of single HA-tagged... that HA frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA is small...researchers used the HA frankenbody to successfully track nuclei in a developing zebrafish embryo over an ...

...LanthaScreen) (Degorce et al., 2009), measuring intracellular calcium (Miyawaki, Griesbeck, Heim, & Tsien,...Biarsenical labeling reagents If you want to label intracellular proteins covalently with FRET acceptors, then... nonfluorescent until they bind a 6-residue tetracysteine tag (sequence: CCPGCC) that can be inserted ...interest, resulting in a C-terminal fusion of a tetracysteine tag. In addition, since these tags are so small...large ion channels involved in skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF...et al. "Hairpin structure of a biarsenical− tetracysteine motif determined by NMR spectroscopy." Journal...

...reverse tetracycline-controlled transactivator) is normally inactive and cannot bind the tetracycline response... inducible promoters. The positive inducible tetracycline ON (Tet-On) system, a versatile tool developed...response elements (TRE) in a promoter. Tetracycline and its derivatives serve as inducing agents to allow...Addgene.org: Inducible Promoters for Bacteria Tetracycline (Tet) On Expression Systems at Addgene Cre-lox...

...by titration with different amounts of tetracycline. Tetracycline regulation is reversible (when the drug...with more elements of control. Tet promoters Tetracycline-dependent promoters are engineered by placing...constitutively silenced proteins. Addition of tetracycline or one of its analogs will induce these transcriptional...transcriptional level.     Fig. 2 Tetracycline on/off promoters and modes of action Check out...

... by ultracentrifugation (75,000 - 225,000 g for 1.5 – 4 hours at 4 °C). After ultracentrifugation, virus...vectors) you can concentrate your virus by ultracentrifugation of the viral stock, followed by resuspension...packaging cell debris before proceeding to ultracentrifugation to avoid contaminating your transduction ... to concentrate a virus solution without ultracentrifugation, you can reduce the volume of cell culture...

...use the autofluorescence extraction feature. Although autofluorescence extraction is an optional feature...correct for this, compensation has to be applied to “subtract” this spillover and correctly distinguish between...possible.  Spectral flow allows for autofluorescence extraction Cells produce a low level of fluorescence called...cytometers include a tool called “autofluorescence extraction” which allows the user to use the autofluorescence...

...enables single-cell anatomical study and neuronal tracing, aka determining where a neuron’s axons and dendrites...labeling so that a cell’s projections are easy to trace and not lost in a jumble of fluorescent neurons.... But notice in figure 4b how difficult it is to trace individual cell’s axons, particularly the two green... as tools for basic science research (neuronal tracing and morphologic studies), as well as translational.... These capsids are useful tools for neuronal tracing and morphology studies when used as part of a two...

...before bleaching. The amount of information one can extract from a biological sample is wholly dependent on...budget, which determines how long and precise we can track individual molecules. Poor photon budgets are a ... couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical fluorophores...FlAsH, ReAsH) and a short genetically encoded tetracysteine (Cys4) peptide tag could be used to label proteins...fluorophores are making chemical dyes increasingly attractive and accessible to cell biologists—and we are ...

... cause cells and muscles to contract depending on the context. Contraction is critical for processes such...1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene called "spaghetti squash...actomyosin cytoskeleton that regulates cellular contraction. This network is composed of actin and myosin...motility, development, cytokinesis, and muscle contractions (think skeletal muscle, cardiac muscle, and ...

...records allows you to track a cell line’s age and monitor any changes. Your tracking system could be as ...just a few publications which have since been retracted due to cell line contamination: Epithelial Mesenchymal...colleague. Time and money are wasted, papers are retracted and there is a loss of reputation that can impede... good recordkeeping, you need to have a way of tracking how many passages the cells have undergone and...

... so, the WHO recommends one strategy: testing, tracking, and social distancing. With countries like South...block and 62°C heat block Approach: Viral RNA is extracted and subject to isothermal amplification by primers...directly using saliva without having to perform RNA extraction but that may work only if there is a high enough...water bath and 42°C water bath Approach: RNA is extracted from a sample and subject to isothermal recombinase...detect SARS-CoV-2 in the samples. First, RNA is extracted and subject to reverse transcription. This is ...

...has become very useful in lineage tracing; “Zebrabow” was used to trace the development of the corneal epithelium... also been applied to cell marking and lineage tracing in other systems, such as zebrafish and Drosophila...Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish. Pan YA, Freundlich...dev.094631. PubMed. Multiplex cell and lineage tracking with combinatorial labels. Loulier K, Barry R,...

...companies which are reliant on contracts with clients is that, when those contracts fall through or are cancelled...(SOP’s) and every item I tested was constantly tracked with barcodes and spreadsheets. It was great to...primary purpose of the company was to fulfill contracts, my work sometimes felt rushed and the focus always...company; new staff hired in anticipation of the contract suddenly have less work to do than was anticipated...