...inhibitory synaptic proteins Fluorescent protein system for C. elegans Fluorescent timers for studying cell... different fluorescent proteins targeting inhibitory or excitatory synapses or that are Cre-inducible,...Split-wrmScarlet, a fluorescent proteins system for Caenorhabditis elegans By Jennifer Tsang Fluorescent proteins...are not fluorescent on their own. When the two fragments come together, they fuse and fluoresce. The team... Addgene hSyn1-SIO-stGtACR1-FusionRed is a Cre-dependent, soma-targeting inhibitory channelrhodopsin....neurotransmitters. Using genetically encoded fluorescent proteins coupled with Fibronectin intrabodies...localize to the synapse and minimize extraneous fluorescence from other regions of the cell. By combining...

... adult human fibroblasts via microRNA-dependent and -independent pathways. EMBO Mol Med. 2017 Aug;9(8)...83. Capecchi Replicating EBNA1 episome Human Fluorescent-tagged EBNA1-mediated expression of human Oct3...and dosage tracking of reprogramming factors Fluorescent tagged episomals for stoichiometric induced pluripotent...induced reprogramming to pluripotency. Stem Cell Reports. 2015 Apr 14;4(4):727-43. Woltjen Plasmid Mouse...Generation of Human Striatal Neurons by MicroRNA-Dependent Direct Conversion of Fibroblasts. Neuron. 2014...self-regulating and nonintegrating viral vectors. Stem Cell Reports. 2014 Nov 6. pii: S2213-6711(14)00307-5. Jaenisch...Cells and B Lymphocytes to Neural Stem Cells. Cell Reports. 2014 Dec 11;9(5):1673-80. Parmar Fibroblasts Astrocytes...

...-light inducible Cre for use in bacteria CRISPR-Casɸ mGold, a new yellow fluorescent protein New CRISPR...expressing an RFP reporter plasmid for recombinase activity were exposed to blue light. Cre-Vvd showed substantially...Mary Dunlop’s lab generated a photosensitive Cre, Opto-Cre-Vvd, by splitting and joining the protein regions...C-terminal and N-terminal Cre fragments together and permitting recombinase activity. Opto-Cre-Vvd is precise, ...light. Unlike other split-Cre systems which can take up to 24 hours to cut, Opto-Cre-Vvd is fast and can cut...introduction & resolution of two DSBs in a fluorescent reporter. Find the plasmids here Yongsub Kim’s ...pAAV-hSyn-Cre-P2A-dTomato is now available in AAV1 and AAV8, as well as AAV5 and AAVrg. Find the Cre-expressing...

...customized fluorescent reporters can be challenging enough, but incorporating multiple reporters into a single...pyralis) reporter plasmids for use in high-throughput screening of pre-mRNA splicing. This reporter system... issues with fluorescent pH sensors, a set of luciferase-fluorophore pH fusion reporters, pRSETb-pHlash...fluorophore is pH-dependent. Signal is simultaneously acquired from the the fluorescent and luminescent ... the 3’ UTR of the reporter mRNA via an MS2 coat protein (NLS-MS2-RFP) the reporter mRNA, which contains...for the fluorescent proteins and an inducible promoter (Plasmid 64543or 64542) When the reporter mRNA is...their reporter mRNAs. Using the TRICK plasmids, Halstead et al demonstrated that 91% of reporter mRNAs...

...cyan-excitable red fluorescent protein (mCyRFP1). Derived from cyan-excitable organge fluorescent protein (CyOFP1...as a donor in FRET (fluorescence resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy...beautifully demonstrate light-dependent Gal4 transcriptional activation and Cre recombination. Find PhoCl ...ancestors), and screened through libraries of new RFP variants for increased fluorescence, brightness, and...phosphatase-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked mCherry...are available at Addgene. Kim, et al. Stem Cell Reports. 2015. PubMed PMID: 25772473 Targeting and manipulating...enhancer elements and combined it with a variety of reporters (i.e. eGFP) and effectors (i.e. DREADDs). The ...

...with that reporter, you can infer its presence through detecting the reporter's fluorescence. Another ...labeling proteins is to fuse them with a fluorescent reporter, such as GFP or mCherry, through genetic...The combination of antibody-fluorophores, fluorescent reporters, and/or dye stains used in your flow cytometry... mentioned previously, you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated.... Next comes our transduction reporter, eGFP. As eGFP emits fluorescence by itself and does not need to...While proteins themselves can emit intrinsic fluorescence when excited with ultraviolet (UV) light, they...to APC. This happens due to a process called Fluorescence Resonance Energy Transfer (FRET).   Figure...

...cleaves the supplied fluorescently labelled ssDNA substrates. Upon cleavage, the fluorescence is no longer quenched...polymerase, recombinase, and the ssDNA-FQ reporters that exhibit fluorescence upon cleavage. Upon amplification...the trans cleavage of the ssDNA-FQ reporter resulting in fluorescence. Cas12a binds all the amplified target...continuous cleavage of the ssDNA-FQ reporter. The resulting fluorescence can be observed under either no ... cleavage of fluorescently labelled RNA, the authors employ a P51 cardboard fluorescence visualizer that...streptavidin portion of the probe via biotin. The fluorescent portion of the probe that is cut will continue...and released when cleaved. Cleavage results in fluorescence and this approach can be incorporated into a...

...expression of Cre-mCherry and a second promoter to drive expression of a Cre-dependent fluorescent CatCH-GFP...CatCH-GFP reporter. Expression of GFP will only result in cells where Cre-mCherry is also expressed. If Cre-mCherry...transgene: an optogenetic reporter, channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The...in photoreceptors and horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion...horizontal cells transduced by both AAVs would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters...different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of the start ...cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences...

...combines the Cre/lox system with optogenetics. Cre activity is controlled by red light by splitting Cre and fusing... Figure 4: The C-terminus of Cre (CreC) and the N-terminus of Cre (CreN) are fused to the truncated Phytochrome... previous Cre/lox or drug-induced Cre systems (ex: tamoxifen). As the name suggests, CreLite combines ...recombine independently and results in a variety of colors depending on the combination of fluorescent proteins...when paired with appropriate fluorescent optics. Existing red fluorescent voltage indicators, however,... dimerize, bringing each terminus of Cre together to restore Cre activity. As soon as red light or PCB...proteins separate, inactivating Cre. This is critical for reducing Cre toxicity in long-term experiments...

...dyes, and fluorescent ligands. Figure created with biorender.com. Fluorescence refers to the emission...stability of fluorescence over time is crucial. For instance, the fluorescence of fluorescein isothiocyanate...the b(right)est fluorescent proteinFluorescent Proteins 101: A History of Fluorescent Proteins  ...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes,...selection and optimization. Fluorescent experiment types: Different types of fluorescent experiments, such as...to the ability to ‘toggle’ between fluorescent and non-fluorescent states in response to a specific stimuli...technologies in fluorescent dyes/ligands Throughout the history of cell biology, fluorescent labeling techniques...

...small molecule activates the fluorescence of the small molecule. Fluorescent RNA aptamers are used to track... applications of fluorescent RNA aptamers in Addgene’s collection. Green fluorescent aptamers Spinach ...better able to maintain its fluorescence intracellularly due to a lower dependency on magnesium and higher... thiazole orange (TO) and fluoresces in the orange or red channel, depending on the structure of the TO...selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution...expression vectors to create a library. Bacteria were transformed, and then sorted by fluorescence-activated cell...vivo studies. Dimerization of Broccoli increases its fluorescence by about 1.8-fold, and may be a better...

... number of fluorescent proteins is steadily increasing. Often, the choice for a fluorescent protein is...refers to the fluorescence intensity of a fluorescent molecule. A higher brightness increases the detected...explains how to critically assess the reported properties of fluorescent proteins, how to do a head-to-head...comparison of fluorescent proteins and how to make a well-informed decision on the best fluorescent protein ...best fluorescent protein for a specific application can seem like a daunting task. Many fluorescent proteins...properties of fluorescent proteins on websites. To make the best choice, the fluorescent protein properties...equipment. Because the performance of a fluorescent protein strongly depends on both the biological system as...

...that’s independent of the reporter. The only caveat is that HA staining is weaker than reporter staining...tagged and instead are conjugated to reporters (i.e. fluorescent dyes, HRP, etc. ) after purification....detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties: HRP, Spaghetti...staining. Picking the right reporter for your RANBody While all three reporters stain cultured cells and ...Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection Method Uses Pros Cons Antigens...antibody and a ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to...proximity thus fluorescently labeling them. HRP RANbody staining results in a ≥10-fold increase in signal ...

...sequences using a fluorescent reporter. In SHERLOCK, a quenched ssRNA fluorescent reporter is added to the...viral genome, and a ssDNA-fluorescently quenched reporter is added. As this reporter is degraded by Cas12a...inactive dCas9 to a fluorescent marker like green fluorescent protein (GFP), creating a customizable DNA...have also led to reduced dependency on specific PAM sequences and the creation of new tools. Figure 3: ...binding signal for Cas9, but the exact sequence depends on which Cas protein you use, and there are several...homology arms). The length of each homology arm depends on the size of the change being introduced; larger...larger insertions require longer homology arms. Depending on the application, the donor DNA may be a single-stranded...

...light which revealed a blue light-dependent shift in mCherry fluorescent to the surface of the bead, suggesting...OptoNB-SOS activated Ras/Erk signaling by using a fluorescent reporter for Erk kinase translocation. These results...protein. OptoNBs were also tagged with an infrared fluorescent protein tag which let the team detect its co-...control protein activity Read about RANbodies, reporter nanobody fusions Learn more about using the secondary...signaling pathway.  As a proof-of-principle, the team created an OptoNB to control the Ras/Erk signaling pathway...

...authors have deposited AAV plasmids for Cre-dependent and -independent versions that are trafficked to the...photostable than fluorescent proteins, making them ideal for single-molecule fluorescence techniques. After...used to measure signal along the dendrite. D) Fluorescence signal as a function of the distance between...pharmacological profiling or interrogation of context-dependent signaling in primary cells. The authors used...types in varying conditions, revealing context-dependent responses and molecular patterns of selectivity...Garcia-Marcos, M. (2024). Direct interrogation of context-dependent GPCR activity with a universal biosensor platform...6E10-A5] in Immunohistochemistry (Human). Addgene Report, doi: https://doi.org/10.57733/addgene.6ncijt.  ...

... are two main classes of reporters: chemiluminescence enzymes and fluorescent proteins. Chemiluminescence...amount of protein you are expecting.  Fluorescent proteins Fluorescent proteins, while more expensive, allow...Nitrocellulose or low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased cost (specialty...without increasing background noise and can reduce the amount of primary antibody required. Reporters There...a wide dynamic range for fluorescent proteins. They do tend to have increased background with higher exposure...Indirect detection Increased time; higher background Chemiluminescence reporter Smaller dynamic ...detection method Increased time; higher background Chemiluminescence reporter Smaller dynamic ...

...elegans expression Other Fluorescent Proteins Localization See full Fluorescent Protein Guide...Selectable markers Viral expression and packaging Reporters, shRNA expression, transgenics and genome... AdEasy®-1 plasmid Back to Top Reporters, shRNA expression, transgenics and genome ...promoter strength pBV-Luc - Luciferase reporter plasmid with very low basal activity shRNA/RNAi...Hynes Lab Plasmids - A system for Cre-regulated RNAi in vivo Dupuy Lab and...mammalian expression, Ligation Independent Cloning (LIC) Bacteria Lac, T7, araBAD p15TV-... from the SP6 promoter pCS2 mt-GFP - Create myc-tag, GFP-tag fusions for transcription...

...break (DSB) mediated homology dependent repair by enhanced green fluorescent protein (EGFP) reconstitution...incubation, you assess the level of fluorescence in your cells under a fluorescence microscope. The higher the ...find your mutant from screening as little as 8-10 pups. The level of fluorescence you saw in the validation...digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR, homologous recombination; SSA, single...containing sgRNA sequences into HEK293T cells. The fluorescence intensity was classified into four groups (4...darker than control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted...your target is inserted, disrupting the EGFP fluorescent signal, for details on this plasmid, see Mashiko...

...mCherry fluorescent protein followed by a stop codon and the coding sequence for a green fluorescent protein...expression of fluorescent proteins and oxytetracycline enzymes. Next, this system was used to recreate natural...representation of cell-free system, capable of producing fluorescent proteins and enzymes. Image from Moore et al....plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE, to identify...designed based on MYC-ChIP-seq data from several MYC-dependent cancer cell lines. It can be applied for identification...different biochemical secondary energy sources to increase protein yields from 36 μg/mL to 266 μg/mL. The...