...non-radiatively transferred from an excited donor fluorophore to an acceptor. Since the transfer of energy...energy does not occur by emission of a photon, the acceptor molecule is not required to be fluorescent. A...

...EF1a/nEF GFAP and variants Synapsin TBG Other Fluorophore/Tag Green Red None Other Cre-dependent color ...non-cre-dependent) Clear Filters ID Name Promoter Fluorophore/Tag Activity Serotype PI 37825 AAV-CAG-GFP CAG...

...preparations are subjected to polyacrylamide gel electrophoresis (PAGE) followed by silver staining or SYPRO...purified and non-purified AAV subjected to gel electrophoresis. Viral capsid proteins VP1, VP2, and VP3 are...

...gRNA Presence of a selectable marker (drug or fluorophore) Delivery method Expression System Components...identify DNA fragments. PCR amplification and gel electrophoresis (for HDR or NHEJ): For large deletions or insertions...

...configuration have been developed, including additional fluorophores, excitatory and inhibitory opsins , genetically-encoded...H134R)-eYFP Vcre Dual recombinase-dependent: Fluorophores Addgene ID Plasmid Logic Sites and Mutations...

... Conversion of Adult Mouse Liver Cells and B Lymphocytes to Neural Stem Cells. Cell Reports. 2014 Dec ...Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells. Cell Stem Cell. 2015 Nov 5;17...

...wavelengths of emitted photons are higher than those of absorbed photons. When photons excite electrons of... you'll need to pick a fluorophore for each target and ensure the fluorophores, together, are able to ... about 380–700 nm. Photons of lower wavelengths are higher in energy, while photons of higher wavelengths...whereby a photon is emitted. This step is called emission. During excitation, some of the photon's energy...the energy of an emitted photon is lower than the energy of the absorbed photon. Correspondingly, wavelengths...outlining a few commonly used fluorophores and their emission spectra. Some fluorophores carry their emission ...and RB545. Fluorophores can also come in combination of two conjugated single fluorophores, like APC-Cy7...

...design utilized Magnet photoswitchable proteins derived from the fungal photoreceptor, Vivid (VVD, N. crassa...split fusions and generate a photoactivatable nickase and a photoactivatable repressor (dCas9). The activity...Off Genome Editing Single-chain Cas9 photoswitches Photoactivation of Cas9 can also be achieved using single-chain...chemistry to photocage CRISPRs The aforementioned techniques each employed a photoactive strategy which...Comparison of photoactivatable CRISPR strategies Publication System nickname Photoactivatable moiety Cas9...et al., 2016 CRISPR-plus Photocleavable ssDNA oligonucleotides Cas9, photocaged-gRNA Gene editing Manna...optogenetic techniques to develop a variety of photoactivatable CRISPR tools. These tools allow scientists...

...proteins predicted to be phosphorylated on at least one site. Phosphorylation is particularly important...predict. Slower Dephosphorylation Rates: FRET-based reporters can have slower dephosphorylation rates than ...by FRET reporters having phosphorylated conformations that are less phosphatase-accessible. Kinase Translocation... signal (NES), both which are phosphorylatable. Prior to phosphorylation, the KTR localizes to the nucleus...biosensors Learn about using phosphoserine to study protein phosphorylation Additional Resources on Addgene.org...specific spectral properties that limit which fluorophores can be multiplexed together. Challenging  to...nucleus, but following phosphorylation bNLS activity is suppressed and NES activity is enhanced, leading...

...fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation microscopy is a fluorescence...decypher cellular dynamics in lymphoid organs. In lymph nodes, for example, two-photon microscopy allowed scientists...worthwhile to test the photostability of your fluorescent protein when using 2-photon microscopy as this ... is advantageous when compared to photoactivable or photoconvertible proteins, as it enables tracking ...mobility with movement from the bone marrow and other lymphoid organs to specific tissues where they can scan... good tools to directly follow immune cells in lymphoid organs. The development of cell sorters together...several labs were able to decipher the journey of lymphocytes from the bone marrow to the thymus where they...

...brightness, photostability, and most importantly, a photoswitching ability. This photoswitching refers to...exciting the fluorophore at peak wavelength can also decrease the rate of photobleaching. Interaction ...function in live cells. Fluorophore properties: The specific properties of the fluorophore, such as excitation...fluorescence quantum yield, the ratio of photons absorbed over the photons emitted. For example, DAPI is much...Ghods, R. (n.d.). Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568-and Fluorescein...ligands Figure 1: Example structures of fluorophores, fluorescent dyes, and fluorescent ligands. ...refers to the emission of light by a substance (fluorophore) that has absorbed light or electromagnetic radiation...

...image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two drawbacks... leads to more rapid photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict...can lead to photobleaching when acquiring multiple images over a large axial range. 2-photon imaging, which...University. No matter whether you are a sports photographer at the Super Bowl, a medical technologist taking...background. In addition to this, the use of multiple fluorophores can add a second layer of contrast, color contrast...further exploit the unique characteristics of fluorophores to block out of focus fluorescence from reaching...large number of optical slices are acquired all fluorophores throughout the sample are constantly exposed...

...non-radiatively from an excited molecular fluorophore (the donor) to another fluorophore (the acceptor) by means of ...the two fluorophores used are in close vicinity. Thus, the distance between the two fluorophores and their...yield of the donor (number of emitted photons per absorbed photons) and the extinction coefficient of the... of a fluorophore pair, donor:acceptor stoichiometry, and cross-talk between the two fluorophore colours...microscopy (FLIM) method. Acceptor photobleaching method The acceptor photobleaching method is simple but limited...dipole-dipole coupling." The emission of the acceptor fluorophore can be measured using microscopy techniques. ...interactions within living cells. By coupling fluorophores to proteins, pioneers of this technique were...

...donor fluorophore to the acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the...absorption of a photon 2. Energy transfer from the excited donor directly to the acceptor fluorophore--think of... of it as a virtual photon 3. Relaxation of the acceptor fluorophore back to its ground state by emission...acceptor fluorophores must be compatible, such that the emission spectrum of the donor fluorophore overlaps...emission of a photon with wavelength specific to the acceptor In order for FRET to occur several constraints...with the excitation spectrum of the acceptor fluorophore. Otherwise the energy transferred from the donor...

...Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon Generation Slower...emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus pyralis...Modification Not Required Required Photobleaching/Phototoxicity Not susceptible Susceptible Subcellular...accurate and safe. In the case of a luciferase assay, photon emission is measured resulting from the catalysis...luciferin, ATP and oxygen as substrates. Production of photons by this bioluminescent reporter occurs slower than... result of the different mechanisms to produce photons, chemiluminescent reporters are generally less ...background levels and improved signal sensitivity since photons are simply measured – they are not required to ...

...to identify a fluorophore, spectral flow cytometers use all detectors for all fluorophores. This allows...samples stained with a single fluorophore. They’re required for every fluorophore you use in your panel, whether...conventional flow cytometry, light emitted by a fluorophore is guided to a single detector using a series...flow cytometry uses one detector to detect one fluorophore and therefore one cellular marker. However, emission... emission spectra of certain fluorophores can “spill over” into detectors that are not the primary detector...detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over into the primary...the full spectrum of light to distinguish one fluorophore from another. Instead of using a single (primary...

...overcome these woes with photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by...modification is 3’ phosphorothioate linkage, where a sulfur replaces a nonbridging phosphate oxygen. Other ...light. The photoactivatable counterpart to this system utilizes a gRNA which is photocaged and unable ...switches. Stabilizing gRNA modifications  Sugar phosphate backbone modifications The recognition sequence... have been employed which modify the sugar or phosphate molecules of the RNA backbone to increase stability...coupling several of them together, for example phosphorothioates with 2’-O-methyl modifications, proved more... specifically ones which do not contain a 5’ triphosphate in the gRNA, reduce the innate immune response...

... conjugated to a fluorophore, not every marker will have a corresponding fluorophore-conjugated antibody...cases, fluorophore-conjugated antibodies may exist, but only with a limited selection of fluorophores. If... tag but not a fluorophore. In a separate step, cells are incubated with a fluorophore-conjugated streptavidin...wide range of fluorophores, allowing for great flexibility when it comes to fluorophore selection for ...common method is a cell surface label, where fluorophore-conjugated antibodies directly bind to epitopes...employ beyond simply incubating cells with single-fluorophore-conjugated antibodies. These methods can include...interaction (Figure 1a). This interaction brings the fluorophore in proximity to the target marker, allowing for...

...improved expression of recombinant phosphoproteins Protein phosphorylation is one of the most abundant forms...questions surrounding serine phosphorylation by adding this phosphorylated amino acid to the genetic code...the original or improved phosphoprotein synthesis reagents. One phosphoserine (SP218) insertion was moderately...plasmids required to make singly or multiply phosphorylated proteins (B40 OTS and pCRT7-GFP). To demonstrate...activating kinase 1 (MEK1) with either one or two phosphoserine residues cotranslationally inserted in their...moderately enhanced while two phosphoserine insertions (SP218/SP222) was dramatically enhanced with the...Addgene’s information page for the Rinehart phosphoprotein system, which includes detailed protocols provided...

...releases energy as a photon. The photon energy is transferred to the acceptor fluorophore, a circularly permuted...range of mitochondrially targeted photoactivatable and photoswitchable FPs that are activated by UV light...subpopulations within the same cell; by photoswitching PSmOrange and photoactivating PAGFP in separate subpopulations... fluorescent pH sensors, a set of luciferase-fluorophore pH fusion reporters, pRSETb-pHlash and pcDNA3.1.... In the case of pHlash, the signal from the fluorophore is pH-dependent. Signal is simultaneously acquired...e43072. doi: 10.1371/journal.pone.0043072. Photoswitchable tools for spatial and temporal control of cell...Three years ago, Brian Kuhlman’s lab designed photoswitchable dimers by fusing the SsrA peptide with the ...