... the summer of 2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C....elegans even easier - now, one can generate a fluorescent protein fusion, transcriptional reporter, and...self-excising cassette (SEC). To make it possible fluorescently label proteins in C. elegans, they inserted ...inserted the SEC into a synthetic intron between a fluorescent protein (FP) and a 3xFLAG tag (see construct schematic...located in this synthetic intron, and only the fluorescent protein and 3xFLAG-tag sequences are inserted...serve as the repair template plasmid to create fluorescent and 3xFLAG-tag fusion proteins. The repair template...the Roller phenotype. These animals contain a fluorescent protein fusion for the targeted gene. After initial...

...New work from Konermann et al. and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in...proceeded to study orthologs from Eubacterium siraeum (EsCas13d) and Ruminococcus sp. (RspCas13d), while Konermann...Ruminococcus flavefaciens (nicknamed CasRx), as well as EsCas13d. Find the Yan et al. plasmids here Find the Konermann...Like other Cas13 enzymes, the Cas13d orthologs described in these papers can independently process their... high targeting efficiency, both of which are described below. Yan et al.: WYL-domain-containing accessory...accessory protein WYL1 increases RspCas13d and EsCas13d cleavage activity The majority of Type VI-D loci...degradation by RspCas13d. RspWYL1 also increased EsCas13d activity, indicating that WYL domain-containing...

...genetically encoded fluorescent kinase reporters. A KTR is comprised of a fluorescently-tagged substrate ...flexible swapping of fluorescent proteins and multiplexing is simple when fluorescent proteins with limited...Multiplex: FRET reporters are comprised of two fluorescent proteins with very specific spectral properties...The relative ratio of cytoplasmic vs. nuclear fluorescence serves as a proxy for kinase activity.  Advantages...about photoswitchable kinases Learn more about fluorescent biosensors Learn about using phosphoserine to...Resources on Addgene.org Find FRET plasmids Find fluorescent biosensors ...

...Zimmer, whose previous books focused primarily on fluorescent proteins and their applications, tackles much...recent resurgence of “Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing... science.”  The three chapters in this section describe the challenges of trusting science in a “fake ... of the book, called simply “Old Science,” he describes the now infamous tale of Rosalind Franklin and...middle-aged scientists, the identification of Green Fluorescent Protein (GFP) and its development into an essential...changed significantly over the last decade. He describes the detection of gravitational waves--a story ...collaboration from scientists around the world. The paper describing the waves’ detection had over 1,000 authors. ...

...you can visualize the protein via fluorescence or chemiluminescence, precipitate the protein out of solution...allows the protein to be detected by chemiluminescence or fluorescence. Western blots are frequently used... a mixture of cells. The technique of using fluorescently-labeled antibodies to detect proteins is referred...antibodies to bind to intracellular proteins. The fluorescence signal is detected by the flow cytometer and...flow cytometry is the ability to use multiple fluorescent signals (up to a dozen or more!) to measure the... fluorophore-conjugated antibodies (i.e., immunofluorescence), or by a colorimetric reaction facilitated...by an enzymatic reaction. Figure 5: Immunofluorescence staining for CD31 (green), CARD14 (red), ...

...direct fusion of fluorescent proteins (FPs) to a yeast gene of interest. These fluorescently tagged genes ...several ready-to-use, modular plasmids, combining fluorescent tags, epitope tags, protease sites, and selection...These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5, ...marker. Lee et al.(1) assessed many of these fluorescent tags in S. cerevisiae, looking at their performance...possible combination of protease site (TEV or PreScission), epitope tag (12xHis, 2xStrep, 3xFlag, Protein...former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide variety...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...

...incubation, you assess the level of fluorescence in your cells under a fluorescence microscope. The higher the ... homology dependent repair by enhanced green fluorescent protein (EGFP) reconstitution. (a) Scheme of ...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...the amount of fluorescence, the better that gRNA is at causing a double stranded break at the target site...screening as little as 8-10 pups. The level of fluorescence you saw in the validation assay should give ...

...laser impacts the magnitude of FSC, SSC, and all fluorescent stains. The cytometer's software will allow you...SSC-H, or FSC-W/SSC-W, respectively. H, A, and W describe the shape of a histogram, which graphically represents... of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W results from...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell...depends on cell type and laser voltage. The voltage describes an electric potential that can be applied to the... my analysis. For this measurement, I used a fluorescent dye that can only enter dead cells, while living...interested in measuring a population based on a fluorescent stain, you might be wondering which second measurement...

...al., 2015). To do so, they fused a different fluorescent protein to various Cas9 variants, SpCas9, NmCas9...PP7, or boxB) that each recruits a different fluorescent protein (BFP, GFP, or RFP) to the genomic loci...to the small molecule, DFHBI-1T to induce its fluorescence. They found that aptamer insertion in the gRNA...8XMS2 which will recruit the HaloTag with red fluorescent ligands to the loci of interest. Dual color detection...sgRNA-Sirius-4X(MS2-PP7)) that result in yellow fluorescence at the loci of interest, the team could visualize...4X(MS2-PP7) showing recruitment of different fluorescent proteins. (B) CRISPR-Sirius used to visualize...loci, what makes CRISPR-Sirius unique? Unlike fluorescent in situ hybridization, CRISPR-based visualization...

...DNA Fluorescence dyes Another way to quantify DNA would be to use fluorescent dyes that fluoresce when...and their corresponding fluorescence. That way, you can compare the fluorescence of your sample against...sequencing. Unlike absorbance-based methods, fluorescence-based methods require a standard curve, a set...These fragments are quantified over time using a fluorescent dye that intercalates into the DNA such that ... using capillary electrophoresis which shows fluorescence units over time. Smaller fragments migrate more...

... the active site. Dronpa also produces green fluorescence when exposed to 500 nm light.  Under 400 nm ... kinase translocation reporter with a mRuby2 fluorescent reporter (ERK KTR-mRuby2). The ERK KTR is a ...active, it phosphorylates the ERK KTR and red fluorescence localizes to the cytoplasm. When MEK1 and ERK...inactive, the ERK KTR isn’t phosphorylated and red fluorescence can be found in the nucleus. For testing kinase...Addgene Blog Learn More about Photoactivatable Fluorescent Proteins Control Protein Expression with Cre-lox... KTR reporter plasmids Find photoactivatable fluorescent proteins in empty backbones in the Michael Davidson...Photoswitchable Fluorescent Proteins ...

...observed with fluorescence microscopy. This application of AHA labeling is known as fluorescence noncanonical...localization of protein synthesis. Alternatively, fluorescent protein tags can be used to facilitate the visualization...protein synthesis in situ is the knock-in of fluorescent tags into the gene locus of interest. An advantage...covalently links the azido containing AHA to a fluorescent alkyne tag. The newly synthesized and tagged ...amplification. The amplified DNA segment is labeled with fluorescent probes that enable visualization of the location...PMC4121734. Additional Resources on the Addgene Blog Fluorescent microscopy techniques - Which is best for me?...strategies Additional Resources on Addgene.org Fluorescent protein plasmids Use FRET to study protein-protein...

... for awards to early career scientists using fluorescent proteins in their research. These awards help...roles in the development and dissemination of fluorescent protein tools. This year, we’ve made some changes...-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3) to study glutamate, a signaling...disease,” says Aggarwal. This biosensor changes fluorescence based on the release of glutamate from neurons...to the roGFP2 cysteine thiols and change its fluorescence properties. The biosensor can also be used along... GCaMP7, measuring calcium signaling related fluorescence during morphine administration and withdrawal...agonist inhibits the neuron (via hM4D). She fused fluorescent protein tags to the receptors to monitor their...

...Resources Cre-lox Resource Guide Fluorescent Protein Resource Guide Fluorescent Protein Blog Posts ...GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies, the project really...constructs similarly to GFP, but commonly used red fluorescent proteins dsRed, mCherry, and TdTomato do not ...GFP and derivatives GFP and YFP, but not red fluorescent proteins, as seen with T-DDOGs. Like T-DDOGs...utliized as scaffolds, including the set of red fluorescent proteins. The T-DOGGs and Cre-DOG are available... C.Y., et al. “A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene ... Caussinus, E., Kanca, O., and Affolter, M. “Fluorescent fusion protein knockout mediated by anti-GFP ...

...and/or fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples...Non-destructive methods, most popularly with a fluorescent reporter, directly couple silencing of your gene... that contains a target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore...have readily observable phenotypes quantified. Fluorescent reporter-based RNAi constructs have been designed...presence of the RNAi transgene, followed by a fluorescence readout of RNAi activity. As a plant researcher...subpopulation can be easily achieved through fluorescence-activated cell sorting (FACS). This is not possible...visually identified as undergoing RNAi through the fluorescent reporter and manually extracted. This process...

...ensure complete sterilization as mycoplasma can even escape 0.2-micron filters. Figure 1: Contamination...you can use PCR based detection, DNA staining, fluorescent tagging, agar plating, etc. Be wise to choose...sensitivity but low specificity (Degeling, 2012). Bioluminescence assay kits are available from different biotech...which trigger cellular mechanisms that generate luminescence. This can then be detected by a luminometer.... to detect mycoplasma. When observed under a fluorescent microscope, the mycoplasma DNA appears in the...small clusters, apart from the cellular DNA. Fluorescent DNA staining is another DNA staining alternative...contamination by other bacteria or extracellular fluorescent signals produced by these bacteria can hinder...

...improved fluorescent proteins through mutagenesis. Chapter 6 - The future of FRET and fluorescent protein... addition to creating and sharing many great fluorescent protein tools, Professor Goedhart is very active..., characterizes and uses genetically encoded fluorescent probes. You can follow him on twitter: @joachimgoedhart...Additional Resources from the Addgene Blog Visit our Fluorescent Proteins featured topic page Read Joachim's Series... Series on Choosing the B(right)est Fluorescent Protein Plasmids 101: Visualizing Subcellular Structures...and Organelles Resources at Addgene.org Find Fluorescent Protein Plasmids Find Plasmids for FRET Find ...

...While proteins themselves can emit intrinsic fluorescence when excited with ultraviolet (UV) light, they... of labeling proteins is to fuse them with a fluorescent reporter, such as GFP or mCherry, through genetic...its presence through detecting the reporter's fluorescence. Another way to label proteins is through treating...cells. The combination of antibody-fluorophores, fluorescent reporters, and/or dye stains used in your flow...to APC. This happens due to a process called Fluorescence Resonance Energy Transfer (FRET).   Figure...brightness of individual fluorophores. Table 1 describes the brightness of a few commonly used fluorophores... As I mentioned previously, you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated...

...with Rosella, a pH-sensitive fluorescent biosensor A pH-sensitive fluorescent biosensor-based assay system...recently deposited with Addgene by Dr. Mark Prescott. Dr. Prescott and Dr. Rod Devenish (both at Monash University...assay. In their recent publication, Kroeze et al. describe their enhanced TANGO arrestin recruitment assay...genome-engineering endeavours of unparalleled complexity in Escherichia coli, like the construction of a so-called “genomically...DsRed is relatively pH-insensitive, while SEP fluoresces at cellular pH (7.0) but not at vacuolar pH (...environments that inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific...When used in combination with their previously described SpyTag/SpyCatcher pair, multiple protein fusions...

...Stiles MA (2022) Improved fluorescent signal in expansion microscopy using fluorescent Fab fragment secondary...101: Introduction to Immunofluorescence  Antibodies 101: Multiplex Immunofluorescence Antibodies 101: Affinity...workings of the synapse by conventional light or fluorescence microscopy (Dani, et al., 2010). How do you ...the resolution barrier while still utilizing fluorescence microscopy, is another option. However, most...antibodies, chemical dyes, and genetically encoded fluorescent proteins (Gambarotto, et al. 2019; Tillberg, ... of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat Biotechnol 34:987...