...and fluorescent reporters being especially popular. We’ll give you an introduction to fluorescent biosensors... of fluorescently-labeled components Fluorescence-activated cell sorting (for cells); fluorescent HPLC...Robert Campbell’s lab display calcium-dependent increases in fluorescence of up to 11,000-fold. FRET biosensors...detects. This change then generates a reporter signal. Reporter signals may be electrochemical or light-based...Picking your reporter Don’t forget about the other end of the biosensor: which fluorescent protein is most...you use to detect your fluorescent protein? The type of detection method depends on what type of information...cameleon” calcium sensors created by Roger Tsien’s lab contain two fluorescent proteins (a FRET pair) separated...

...dimerized by the GBPs/GFP. The new system, CRE-DOG (Cre Dependent On GFP), is activated by GFP and derivatives...Addgene Resources Cre-lox Resource Guide Fluorescent Protein Resource Guide Fluorescent Protein Blog Posts...Although Cre isn’t a modular protein, Tang et. al were able to create a split version of Cre that is only...transcription or Cre recombinase activity only in the presence of GFP. These systems, known as T-DDOG and Cre-DOG,...a UAS-driven luciferase reporter construct. In the absence of GFP, no reporter output was observed in ... and a reporter construct into the mouse retina. GFP expression robustly activated the reporter gene TdTomato...Podcast Interview with Connie Cepko Combining Cre and GFP with Cre-DOGs The T-DDOG results showed that GFP ...

...Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically encoded fluorescent kinase...Multiplex: FRET reporters are comprised of two fluorescent proteins with very specific spectral properties...together. Challenging  to Design: FRET reporter activity depends on both the distance and the orientation...kinase reporters. A KTR is comprised of a fluorescently-tagged substrate for a kinase of interest which is...translocation of the reporter. As a result, the KTR is shuttled in and out of the nucleus depending on its phosphorylation...flexible swapping of fluorescent proteins and multiplexing is simple when fluorescent proteins with limited...they are easy to create and simple to multiplex. Problems with FRET kinase reporters Difficult to Multiplex...

...sensors include syn-jYCaMP1s (slow variant), the Cre-dependent syn-FLEX-jYCaMP1s, and the axon-targeted syn-FLEX-axon-jYCaMP1s...syn-FLEX-axon-jYCaMP1s. Find these AAVs at Addgene Cre-dependent EYFP AAV in the new serotype PHP.V1 that exhibits...this AAV at Addgene New serotypes for the Cre-dependent Flpo recombinase pEF1a-DIO-FLPo-WPRE-hGHpA, ...in this post: Yeast-based protein evolution Fluorescent tools for mammalian cells Nanobody purification...enables tracking of protease activity with a fluorescent protein--no affinity purification required! The...doi.org/10.1038/s41592-019-0665-7 Optimizing fluorescent tools for labeling structures and compartments...mammalian cells By Angela Abitua Genetically encoded fluorescent proteins are often used to label  specific structures...

...and fluoresce. FlipCherry: Red fluorescence protease reporter To add to the fluorogenic reporter toolkit...genetically encoded fluorescent executioner caspase reporters. Several apoptosis reporters have been developed...results in a change in fluorescence emission. FRET and fluorescence based reporters however have several...name FlipGFP. FlipGFP increases fluorescence 100-fold compared to other reporters (Zhang et al., 2019)....be applied to other fluorescent proteins to create an array of multicolor reporters that will enable imaging...transfer (FRET)-based executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase...necessary for fluorescence. When these parts are separated from each other, the protein cannot fluoresce. When...

...rate depends on power is a property of the fluorescent protein. This can differ between fluorescent proteins...desirable to have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may... the ideal situation, a fluorescent proteis should emit a stable fluorescence signal, showing no or little...This non-linear dependence on excitation power is important since different fluorescence imaging strategies...measurements for different fluorescent proteins and by comparing how the fluorescence intensity changes over...cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend on ...your understanding of reported data. Photobleaching results in a loss of fluorescence, which, in its simplest...

...bioluminescent reporter occurs slower than fluorescent-based methods, such as excitation of Green Fluorescent Protein...Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as luciferase, usually serves...photons, chemiluminescent reporters are generally less bright than fluorescent proteins, but have the advantage...luminescence versus fluorescence.  Table 1: Properties of Luminescence versus Fluorescence   Luminescence... is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green light with a...several studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays...available to choose from) is now a common feature in reporter plasmids.  Learn How Luciferase Can Be Used In...

...background fluorescence and in-frame fluorescence.  Figure 2: The GEmCherry reporter. Image from...assays. Recently scientists have created CRISPR fluorescent reporter assays to quantify targeted Cas9 ...(NHEJ). In these reporters, editing typically results in a change in the fluorescent readout. In this ...this blog we will focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...enrich for mutants through the presence of a fluorescent reporter. Lastly, once a mutant is identified, the...

...  NIR reporters and biosensors NIR reporters can tell us about cellular events via  fluorescence changes...after normalization to fluorescence of co-transfected EGFP. e Originally reported as a monomer16, IFP2.0...proteins provide a scaffold for antigen-dependent fluorescent nanobodies. Nat Methods submitted (2021)...directed evolution approach applied to fluorescent voltage reporters. Nat Chem Biol 14, 352-360 (2018). Yu...sets of near-infrared fluorescent proteins (NIR FPs) and biosensors have been created recently. As developers.... & Verkhusha, V.V. How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian Cells...Addgene. Biliverdin chromophore Near-infrared fluorescent proteins (NIR FPs) were derived from natural ...

...expressed independently) originally fluoresce green or blue and, when they mature, fluoresce red. The ...immature fluorescence from the FP timer is dependent on the total expression time but independent of protein...time.Hallmarks of fluorescent protein timers When FP timers are first expressed, they predominantly fluoresce one ...., et al. "Monomeric fluorescent timers that change color from blue to red report on cellular trafficking...Advice on Choosing Your Fluorescent Protein Resources at Addgene.org Find Fluorescent Protein Timers Browse...Browse All Fluorescent Protein Empty Backbones Check Out Our Fluorescent Protein Guide ...tetrameric fluorescent proteins can be. Cytotoxicity, improper localization, and decreased functionality...

...stranded DNA bound by tTALE-FP with the fluorescent protein fluorescing green. Image from Shin et al., 2019...intercalating fluorescent dyes for DNA-protein interaction studies? Kyubong Jo's lab generated fluorescent protein...Calcineurin reporters allow scientists to measure calcineurin dynamics and can serve as a drug-screening platform...sequences which brings the attached fluorescent protein to the DNA. They created plasmids for purification of...remove a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed...https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article contributed...protein CZI, which in turn binds to calcineurin-dependent response elements (CDREs) and drives gene expression...

...successful screen, it is necessary to identify the gRNAs that elicit the phenotype, and this depends on the...whole organism imaging. They created an endogenous ApoB-NanoLuc reporter by inserting the NanoLuc luciferase...Cell, 2019. PubMed PMID: 30639098. LipoGlo: A reporter for measuring ApoB bound lipoproteins Article ...luciferase downstream of the apoB gene. Using this reporter, the researchers explore many aspects of ApoB-...2019. Pubmed PMID: 31366908 Adapting sigma54-dependent transcription for CRISPR activation in bacteria...However, the promoter in question must be σ54-dependent and the system may not be adaptable to distantly... systems and further the study of natural σ54-dependent promoter systems. Find these CRISPR activation...

...and its cre-dependent version at Addgene! Fluorescent protein and control AAV These fluorescent protein...DreO-bGHpA 121675  AAV9  pAAV-EF1a-fDIO-Cre (FLP-dependent CRE) 55634  AAV1  pAAV-EF1a-mCherry-IRES-...constitutive (pAAV-Syn-Archon1-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2...exhibits high contrast between peak fluorescence and resting fluorescence. It is useful for activity imaging...exhibits high contrast between peak fluorescence and resting fluorescence.  It is useful for activity imaging...pGP-AAV-syn-FLEX-jGCaMP7b-WPRE) exhibits the brightest resting fluorescence of the GCaMP7 family of calcium sensors and ...the endoplasmic reticulum, but exhibits larger fluorescent responses to GABA. Find the iGABASnFR and its...

...RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence suggests an increase in autophagic...flux, while an increase in GFP fluorescence relative to RFP fluorescence suggests a decrease in autophagic...autophagy. The development of several fluorescent autophagy reporters now allows for the measurement of autophagic...to lysosomal proteases is key to its pH-dependent fluorescence. In neutral environments, dimeric Keima...Rosado, Carlos, et al. "Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and...mammalian cells using a single fluorescent biosensor." Scientific reports 5 (2015): 12397. PubMed PMID:...photoconversion Light sheet fluorescence microscopy Resources on Addgene.org Fluorescent protein plasmids and...

...fundamentally the reporter construct monitors the expression of a nearby gene. The reporter gene serves as...any way. For example, if green fluorescent protein (GFP) was the reporter gene and green cells were observed...gene. The actual reporter construct can be something phenotypic (such as a fluorescent protein or something...introduce reporters throughout an organism’s genome. These reporters have historically been used extensively...stimulate the expression of the reporter. The system is made up of a reporter gene flanked by a minimal promoter...isolated and screened. Depending on the marker, traps can be used as high-throughput screens to introduce...promoter – all rely on the random integration of a reporter construct in close proximity to an endogenous ...

...NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...better biosensors. NanoLuc®-fluorescent protein fusions for imaging Fluorescent proteins are fantastic imaging...more penetrative orange fluorescent protein fusion (OgNLuc). Both fluorescent proteins can be excited ...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged...for infection of host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected...cyan, yellow, green, orange and red fluorescence. The paper reports a wide variety of applications of the...transfer of energy from one fluorescent protein to a compatible fluorescent protein. FRET can be measured...

...available. Detection methods Many calcium reporters use a single fluorescent protein (FP), requiring only a simple.... This sensor switches from green fluorescence to a red fluorescent state only by simultaneous (1) user-controlled...expression, and compatible with constitutive or Cre-dependent expression. These sensors have also been re-...Genetically encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium binding proteins (... (e.g., calmodulin or troponin C). Their fluorescent readouts are used to monitor calcium concentrations...GCaMP6 family of sensors are commonly used calcium reporters with different kinetic parameters. There are three... of the sensor, and (3) subsequent change in fluorescence only occurs at high calcium concentrations. ...

...CRISPRa depended on continued expression of dCas9 and gRNAs whose expression varies depending on transgene...construct and the fluorescently-labeled gRNA multiplex. Read the paper in Scientific Reports Find the plasmids...The new editors reported in this study have ~10-100-fold lower average Cas9-independent off-target DNA ...DNA editing, ~5–50-fold lower levels of Cas9-dependent off-target DNA editing, and maintain ~50–90% of average...stem cells Lindy Barrett’s lab developed new fluorescently labeled piggyBac vectors to deliver uniform ... lines with AAVS1-integrated, inducible, and fluorescent dCas9-KRAB and dCas9-VPR for quantifying and ...cost-effective screening methods, David Liu’s lab identified and validated CBEs with reduced Cas9-independent deamination...

...artifacts. If you're using green fluorescent protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional...strategies can help you make better fluorescent fusion protein reporters for your studies and greatly lower... second FP, often decreasing the fluorescent signal from the first FP and increasing the signal from the... transcriptional reporter or as a general cytoplasmic label of cells, there's no problem. You're OK. However... means that your POI fused to GFP or another fluorescent protein (FP) could be forming dimers in cells... a particular type of molecule to form a dimer depends on its molecular affinity, termed the dissociation... Figure 1: Each problem with dimerizing fluorescent proteins is illustrated. 1) Membrane proteins...

...mouse strain in which Cre expression causes the induction of both DsRed fluorescence and GFP shRNA. Mice...level. Upon Cre/loxP recombination, each transgene expresses one of three possible fluorescent proteins,...This system uses two plasmids in tandem, with Cre-dependent inversion determining between 0 and 2 FPs expressed...Cas9 gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the ... recombines independently, generating tens or hundreds of possible combinations (depending on the number...mutagenesis libraries or to insert heterologous genes, reporters, or other markers into a target genome. They did...engineer a wide variety of GFPs with diversity in fluorescence brightness, intensity, excitation and emission...