...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...

...enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In...ligation product in a gel, but we can now use fluorophore- quencher pairs or qPCR-based methods to greatly...downstream side (the base pair providing the 5 ́-phosphate to the ligation). The structural/mechanistic reason...the reaction. This “peeling back” of the 5 ́-phosphorylated base can be observed in the crystal structures...identifiable and quantifiable by capillary electrophoresis (CE) (see below). This method allowed screening...gene PCR-ligase detection reaction-capillary electrophoresis assay." Journal of clinical microbiology 45.6...mixed population of excess wild‐type DNA." Electrophoresis 34.9-10 (2013): 1415-1422. PubMed PMID: 23463388...

...scientist and photographer based at the University of Queensland, whose outstanding marine photography has attracted...” Daniel went on to discuss his work: “Macro photography has been my passion for a while - a tool to expand...life cycle of the creature they deal with.” So photography can be used as a way to better understand and...scientist and non-scientist alike. “The other thing photography has shown to me - it requires an awful amount...data, publish in time, defend the thesis, etc. Photography is critically important in many fields of biology... can be found on his website http://www.microworldsphotography.com/. Another artist who spent his life...artists can compete in the Nikon Small World Photomicrography Competition - a 40 year old international ...

...mEGFP (Zacharias et al., 2002). GFP needs oxygen Chromophore formation requires a fully folded beta-barrel...an intramolecular reaction that generates the chromophore (Tsien, 1998). Importantly, this reaction requires...-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a protonated and a deprotonated... bind specifically and with high affinity to fluorophores. Examples are the FlAsH tag, the His tag and...enzyme to covalently label the peptide with a fluorophore. Both systems are a hybrid between a genetically...homology domains can be used to detect specific phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production...Péter, and Tamas Balla. "Live cell imaging of phosphoinositides with expressed inositide binding protein domains...

...yourself or you could “clone by phone”. One of my professors was excellent at phone cloning with labs around ... NanoLuc® Luciferase from a rather dull 19kDa Oplophorus luciferase by Promega’s Advanced Technologies...problematic because excitation light can cause phototoxicity, autofluorescence, and perturb biological phenomena... molecules. FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting...exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing...sensor with the following characteristics: No photoexcitation High Ion specificity High sensitivity Easy ...and colleagues (8) were interested in using photoactivatable, optogenetic accuators to depolarize or hyperpolarize...

...spectrum (hence the name), which is due to a chromophore formed from a maturation reaction of three specific...surprising aspects of GFP was the fact that the chromophore forms spontaneously and without additional co-factors... β-sheet-containing “barrel” shape, with the chromophore concealed at the center of the structure, shielded...that increased the fluorescence intensity and photostability of GFP. This also shifted its major excitation...been introduced to GFP and new iterations of fluorophores are constantly being engineered. Table 1 lists... 1: The specific mutations comprising common fluorophores Fluorescent Protein Mutations Relative to...Known Function S65T Increased fluorescence, photostability, and a shift of the major excitation peak to...