...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 ...

...Alkaline phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline phosphatase. Normally...accumulation of Pho8Δ60 in the vacuole, where it is cleaved to generate an active enzyme. Pho8 phosphatase activity...protein. To monitor autophagy, the Pho8Δ60 mutant protein is expressed. Pho8Δ60 localizes to the cytosol in ...Normally PHO8 is synthesized at the ER, delivered to the vacuole via the secretory pathway, and then cleaved...activity or the molecular weight shift of Pho8 from its uncleaved to cleaved form is the final read out of...’s slow and only measures bulk autophagy. Morphological assays Electron microscopy: Electron microscopy...identification of autophagic structures based on morphology. Autophagosomes are relatively easy to identify...

... not photostable, and were presented in the study to support the uniqueness of the photostability of the...polymerase III transcription using a photostable RNA–fluorophore complex." Nature chemical biology 13.11...known promoter. Addgene’s Plasmids 101: Aptamer Fluorophores describes the enrichment process used to evaluate... and non toxic to cells. Because it rapidly photobleaches, DFHBI can only be used for qualitative, but...hydroxybenzylidene-imidazolinone-2-oxime (DFHO), a fluorophore modified from DsRed. DFHO shares many structural... Corn and its small molecule makes Corn more photostable than Spinach and Broccoli, and useful for quantitative...increased brightness compared to Mango, and all are photostable and useful for both live and fixed cell imaging...

...FSC/SSC), the detected photons can be represented through a graph of photocurrent vs. time. The resulting...tumor-infiltrating lymphocytes of that tumor. In Figure 3 you can see how I gated out my lymphocyte population...(SSC). FSC and SSC depend on the cell’s size, morphology, and structural complexity.   Height, Width...your individual experiment. Practical example: lymphocytes in a mouse tumor sample You made it! So much ...flow analysis, I can however clearly identify my lymphocyte population (10.7% of all recorded events) based...electric potential that can be applied to the photomultiplier inside the cytometer to increase the electric...case, I and other lab members had worked with lymphocytes before, so I knew where to look for them on the...

...Tichy et al., 2019), photoswitchable tethered ligands (PTL), and photoswitchable, orthogonal, remotely-tethered...pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be used to activate... G-protein coupled receptors (OptoXRs), and photoswitches. Light-gated ion channels directly open and ...potential and a host of other cellular functions. Photoswitches are engineered molecules that induce conformational... special, covalently linked molecules (i.e. photoswitches) attached near the binding pocket of an engineered...activates downstream secondary signaling pathways. Photoswitchable tethered ligands hold the ligand in close proximity....   Controlling secondary pathways with photoswitches OptoXRs are great for activating entire signaling...

... levels of the fluorophore or fluorochrome-stained protein will emit a stronger photon than a cell expressing...emits green, yellow and orange photons while PE emits yellow and orange photons. If FITC and PE were being...cannot be used for subcellular analysis such as morphology or subcellular localization studies. For these...light source, a cell expressing the associated fluorophore or stained with the associated fluorochrome, ...captured by detectors. The strength of the emitted photons varies from cell to cell depending on the level...expression, measured using antibody conjugated fluorophores Fluorescent antibodies get around the challenges...contain a mix of cells including granulocytes, lymphocytes, and monocytes. Due to differences in size and...

...increased brightness, sensitivity, and photostability under two-photon illumination than previous GEVIs. Some...called JEDI-2P (Liu et al. 2022) optimized for two-photon imaging. They use a unique and powerful high-throughput...based on performance metrics specifically for two-photon microscopy. The resulting JEDI-2P has faster response...increased voltage response, brightness, and photostability. It enables sustained deep-tissue imaging in...recording using a fast indicator evolved for two-photon microscopy. Cell. 2022 Aug 16. pii: S0092-8674(...domain of ZAP70 (ZtSH2) which binds only the phosphorylated state of an immunoreceptor tyrosinase-based...only when the RTK is activated and the ITAM phosphorylated. To demonstrate that pYtags can be generalized...

...to our mScarlet Podcast Segment    Photocleavable protein PhoCl expands optogenetics toolbox Increasingly...proteins. PhoCl is a new member of this class that works via a different mechanism - the PhoCl protein ...illustrates the utility of PhoCl in a variety of settings. For instance, inserting PhoCl in between a protein... al., 2007) and includes four promoters, six fluorophores with nonoverlapping emission spectra (N- and...initiation of reprogramming (evaluated by an alkaline phosphatase-positive assay) as well as stabilization (Nanog-GFP...mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet, fluorescent lifetime ...NES, enables control over localization. Inserting PhoCl between an enzyme and an inhibitor domain similarly...

...salts. Capillary electrophoresis Quantifying DNA using capillary electrophoresis is similar to quantifying... using gel electrophoresis. Except, it’s smaller. And automated. Unlike gel electrophoresis, you only ...wavelength that they maximally absorb. The spectrophotometer measures these absorbances using UV-transparent...specific for double strand DNA (as compared to spectrophotometric methods that measure all nucleic acids). ... sample concentration for you. Agarose gel electrophoresis Though not the quickest way to quantify DNA...methods can’t tell you and you don’t need a spectrophotometer or a fluorometer to quantify DNA this way....nanofluidic channel and the sample is separated by electrophoresis. Smaller fragments migrate quicker than larger...

...can hydrolyze phosphatidylcholine (PC), the predominant membrane lipid, to phosphatidic acid (PA) or it...activity. Researchers have designed a variety of photoswitchable elements into many other types of proteins.... of experiments. Opto-RhoGEFs manipulate cell morphology and adhesion But what if you’re interested in...cytoskeleton – actin? Actin dynamics control cell morphology, movement, and more, often driven by Rho family...filopodia formation, cell contraction, or other morphological changes, depending on the specific Rho GTPase...published OptoPLD, an optogenetically controlled phospholipase D (PLD). Now they present a set of variants ... it can swap phosphatidylchonine’s choline head group for other natural or synthetic head groups. The ...

...available now. It is for recombinant expression of phosphoserine-including proteins in E. coli (Zhu et al., 2022... 2022). It uses the TAG codon for the phosphoserine and, because it uses ∆serB strains, does not require...require exogenously added phosphoserine. A community resource Like Addgene, GCE4All is a community resource...shortlisted several protocols, including their phosphoserine ncAA protocol, to optimize and share, they’re..., R. (2022). Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli...

...categories of fluorescent proteins Photoactivatable / Photoconvertible: These proteins can switch their ... fluorescence. Examples for such photoactivatable or photoconvertible proteins are PA-GFP, Dendra2, and...e.g. mCherry). Oxygen: The maturation of the chromophore on many FPs (particularly those derived from ...takes the FP to correctly fold and create the chromophore. This can be from a few minutes after it is translated...0.1) and this should be taken into account. Photostability can be affected by experimental parameters ...excitation light intensity, pH or temperature). Photostability: Fluorescent molecules gets bleached (i.e. ...after prolonged exposure to excitation light. Photostability can be as short as 100ms (EBFP) or as long ...

...absorption of 540 nm and two-photon excitation around 1,040 nm, has larger photocurrents, and is more selective...they found an indicator that is optimized for 2-photon imaging of voltages. This includes monitoring of...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image ...recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). ...channelrhodopsins (KCRs), from a fungus-like protist Hyphochytrium catenoides (Govorunova et al. 2022). The authors...have high selectivity for K+ over Na+ and rapid photocurrent kinetics. They demonstrate the use of HcKCR1...frequencies with (magenta) and without (black) photoactivation of HcKCR1 in mouse neurons. D) Membrane voltage...

...panel.  First, fluorophores vary in brightness. You don’t want a super-bright fluorophore, like PE, on ...assay you can do. Most non-HRP conjugates are fluorophores, which work not by reacting to a chemical, but...then read by a detector panel in the machine. Fluorophores are light sensitive, so antibodies conjugated...before you make your purchase. Picking a single fluorophore for an experiment is easy, but what about those...of interest! Research the brightness of your fluorophores before buying and try to inversely pair brightness...possible.      Fig 2: Try to inversely pair fluorophore strength with protein abundance.    Second...Second, you’ll need to consider spectral overlap. Fluorophores signal are actually quite broad, resulting in...

... As Nano-lanterns are not subject to photobleaching/phototoxicity, they successfully imaged multiple subcellular...long-term imaging, especially where photobleaching and phototoxicity are a concern, these constructs come...Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be affected by...by both autofluorescence and photobleaching. Researchers have long been interested in using luminescence...bioluminescence resonance energy transfer (BRET). Photons emitted by a Renilla luciferase variant are used...in the culture medium to allow RLuc8 to produce photons. The original nano-lantern emitted yellow-green...Resources on the Addgene Blog Learn about Photoactivatable Fluorescent Proteins Check Out Fluorescent...

...together. Common conjugates in the lab Fluorophores Fluorophores allow you to detect your antibodies using...microscopy, flow cytometry, or spectrophotometry. The range of fluorophores available for antibody conjugation...tool for comparing different fluorophores. We also touched on fluorophores as antibody conjugates in an...sure to check the spectral properties of the fluorophores you are considering to make sure that they’ll...equally, if not more, common conjugate class than fluorophores are enzymes. The most common enzyme conjugates...are Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP). Various substrates for each of these enzymes...