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We narrowed to 317 results for: pho

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  1. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    Published
    June 23, 2016, 2:30 p.m.
    ...SDS-PAGE gels containing Phos-Tag, a small molecule that binds specifically to phosphate moieties and promotes... al. "Detection of phosphorylation on large proteins by Western blotting using Phos-tag containing gel...MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic dual...shift in phosphorylated, but not non-phosphorylated proteins (8). Quantifying the ratio of phospho- and non-phospho-bands...of the dephosphorylation reaction can be tracked through the comparison of phospho:non-phospho species...modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array...suggest that phosphorylation is far more prevalent than previously appreciated. PhosphoSitePlus, one of the...

  2. Synthetic Photobiology: Optogenetics for E. coli

    Type
    Blog Post
    Published
    Sept. 8, 2015, 2:30 p.m.
    ...or dark conditions, the chromophore activates the RR (OmpR) via phosphorylation, promoting transcription...histidine kinase/photosensor senses various light inputs, and an obligate chromophore allows the system...dark conditions, the chromophore activates the response regulator via phosphorylation, promoting transcription...in E. coli easier and more robust. Synthetic photobiology: why use light? In synthetic biology and bioengineering...TCSs). TCSs consist of a histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses...two-component system (TCS). Levskaya et al. fused a photosensory domain from cyanobacteria to a common E. coli...hybrid construct senses light, and an obligate chromophore allows the system to respond to various light...

  3. Lighting Up Cell Signaling with Photoswitchable Kinases

    Type
    Blog Post
    Published
    July 25, 2017, 1:20 p.m.
    ...dimerizing photoswitchable Dronpa (dron= a ninja term for vanishing, and pa= photoactivation) domains on... Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to create other photoswitchable kinases? The...Signal transduction pathways are a lot like cell phone networks. Protein kinases deliver messages to the...-specifically activated and, like the game of telephone, often the signal must be transmitted by intermediate... lab present a generic design for creating photoswitchable kinases and use MEK1 as a model kinase. In ...site of a constitutively active MEK1. This photoswitchable MEK1 is called psMEK1. When exposed to 500 ...psMEK1 also worked for creating three other photoswitchable kinases: psMEK2, psRaf1, and psCDK5. MEK2 is...

  4. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    Published
    April 11, 2017, 2:30 p.m.
    ...state contains a fluorophore and a quencher in close proximity. For instance, the fluorophore and quencher...of aptamers means they can be used with or as fluorophores in several different ways. Researchers have ...fluorescent in situ hybridization (FISH) where fluorophore-linked antibodies are used to visualize RNA. ...removing the quencher from the proximity of the fluorophore and subsequently leading to a positive signal...signal. The same effect can be achieved for a fluorophore that relies on dimerization for activation, only...used as powerful biosensors, the bound protein fluorophores and quenchers are still relatively bulky. One...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether...

  5. Seeing Red: Simple GFP Photoconversion

    Type
    Blog Post
    Published
    Aug. 4, 2015, 2:30 p.m.
    ...biological research. Photoconvertible fluorescent proteins Newly developed photoconvertible FPs like PsMOrange...Applications and limitations of GFP photoconversion One concern with photoconverting GFP is the expression level...and efficiently photoconverted in plant, Drosophila, and mammalian cells. Photoconverted GFP is compatible...Davidson MW, Looger LL. A bright and photostable photoconvertible fluorescent protein. Nat Methods. 2009...accidental discovery that laser treatment can photoconvert GFP from green to RED! This simple technique...time-consuming to repeat the process with these new photoconvertible proteins. With Sattarzadeh et al.’s discovery...discovery that GFP itself is photoconvertible, new studies of spatiotemporal dynamics can be undertaken using...

  6. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    Published
    June 8, 2017, 2:30 p.m.
    ...emitted photons before they are destroyed. Factors that affect photostability The photobleaching rate of...will discuss how to select a photostable fluorescent protein. Photobleaching is the irreversible destruction...destruction of a fluorophore under the influence of light. Any fluorescent molecule will photobleach at some point...have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display...likelihood that a fluorophore will bleach. This is, however, not the full story. The photobleaching rate does ... 2-fold does not reduce photobleaching by half. How exactly the photobleaching rate depends on power is...to compare the photostability of different fluorescent proteins? Measuring photostability Since it is clear...

  7. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    Published
    April 25, 2017, 2:30 p.m.
    ...illuminated with UV-violet light (~400 nm), the chromophore photoconverts predominantly to the anionic form, causing...presented first, it causes irreversible photoconversion of the chromophore from the neutral to the anionic form...mEosFP, can form it’s chromophore at a more comfortable 37°C (5). Dendra2 photoconverts from green to red ...reversible PA-FPs (also known as photoswitchers) can be photoswitched from a dark state to a bright fluorescent... for vanishing, and pa, for photoactivation) (6). Dronpa is photoactivated by blue wavelengths of light...consider, such as brightness, photostability, pH stability, chromophore maturation rate at 37 °C, turnover...-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce though...

  8. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    Published
    July 19, 2016, 2:30 p.m.
    ...biophysical interaction between two fluorophores. When the first fluorophore (the donor) is excited, it can...acids/lipids of interest are fluorophore labeled and that these fluorophores can undergo FRET when they ...targeting a FRET fluorophore to a biomolecule is a bit like a Fedex delivery. Your fluorophore package needs... energy directly via resonance to the second fluorophore (the acceptor) if the two probes are close together...physical distance between the donor/acceptor fluorophores (Stryer, 1978), a measurement that is unaffected...label your biomolecule of interest with FRET fluorophores? When is absolute labeling specificity needed...measure it). Molecular mailbox: How to get your fluorophore delivered to the right place? FRET experiments...

  9. Photosensitizer Induced Cell Ablation with FAP-TAP MG-2I-dL5**

    Type
    Blog Post
    Published
    Sept. 19, 2017, 1:20 p.m.
    ...technique! What is a photosensitizer? First, for a few definitions. A photosensitizer is a dye or protein... higher photostability than KillerRed, with KillerRed reaching greater than 75% photobleaching after just...system? Turns out that with light-inducible photosensitizers and a quick zap of the proper color light,...light, you can do just that.  Photosensitizing dyes and proteins have been around for awhile (check out this...Bruchez and Tsang labs recently developed a photosensitizer composed of the protein complex complex and... oxygen species (ROS). The ROS produced by photosensitizers inactivate target proteins or cells while ...having little effect on neighboring cells. The photosensitizer used in this work is composed of two parts:...

  10. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    Published
    Sept. 8, 2016, 2:30 p.m.
    ...called “the photon budget”: each sample has a limited number of fluorophores and each fluorophore can emit...the photon budget and pushing the frontiers of fluorescence microscopy often requires more photons. For...cells can decrease the number of fluorophores, compromising the photon budget. Likewise, the switch from...desperate to extract a few more photons from a sample. Chemical fluorophores can be substantially brighter...more photostable than fluorescent proteins, providing a straightforward way to improve the photon budget...rhodamine fluorophores, such as the Alexa Fluor and ATTO dyes, with improved brightness, photostability, and... of photons for your imaging experiment. Innovative labeling strategies and improved fluorophores are ...

  11. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

    Type
    Blog Post
    Published
    March 19, 2019, 1:31 p.m.
    ...Listen to the photoswitchable protein podcast segment Reversible control of photoswitchable proteins, which...., 2018). They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into a loop...ratio, high sensitivity, responds quickly, is photostable, and is compatible with optogenetic control. ... Discovery of new protein binder tools for photoswitchable proteins cPYP and AsLOV2 Article contributed...temporally control cellular processes in vivo. Many photoactive proteins exist in nature, but developing them... either the light- and dark- states of the photoswitchable cPYP, which changes in conformation in response...bind to only the light- or dark- state of the photoswitchable proteins offers a powerful approach to control...

  12. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    Published
    May 28, 2024, 1:45 p.m.
    ...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...

  13. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    Published
    Sept. 3, 2020, 12:15 p.m.
    ...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...

  14. Measuring Kinase Activity at the Single-Cell Level with Kinase Translocation Reporters (KTRs)

    Type
    Blog Post
    Published
    July 26, 2018, 12:46 p.m.
    ...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...

  15. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    Published
    Aug. 15, 2017, 1:24 p.m.
    ...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...

  16. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    Published
    March 21, 2024, 1:15 p.m.
    ...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...

  17. Which Fluorescence Microscopy Techniques is Best for Me?

    Type
    Blog Post
    Published
    Oct. 10, 2017, 1:57 p.m.
    ...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...

  18. Tips for Using FRET in Your Experiments

    Type
    Blog Post
    Published
    Nov. 5, 2014, 3:41 p.m.
    ...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...

  19. Fluorescent Proteins 101: Introduction to FRET

    Type
    Blog Post
    Published
    June 27, 2017, 1:03 p.m.
    ...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...

  20. Fluorescent Proteins 101: Luciferase

    Type
    Blog Post
    Published
    June 24, 2014, 3:59 p.m.
    ...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 ...

  21. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    Published
    Aug. 3, 2023, 1:15 p.m.
    ...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...

  22. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    Published
    Oct. 20, 2022, 1:15 p.m.
    ...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...

  23. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    Published
    June 27, 2024, 1:15 p.m.
    ... 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...

  24. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...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 ...

  25. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...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...

  26. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    Published
    April 13, 2017, 2:30 p.m.
    ...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...

  27. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    Published
    Jan. 29, 2019, 2:27 p.m.
    ... 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...

  28. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    Published
    April 4, 2024, 1:15 p.m.
    ...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...

  29. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    Published
    June 27, 2023, 1:15 p.m.
    ...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...

  30. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    Published
    July 20, 2021, 1:15 p.m.
    ... 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...

  31. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    Published
    Dec. 6, 2022, 2:15 p.m.
    ...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...

  32. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...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...

  33. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

    Type
    Blog Post
    Published
    July 21, 2020, 1:15 p.m.
    ...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...

  34. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    Published
    Sept. 28, 2023, 1:15 p.m.
    ...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 ...

  35. GCE4All: Making Genetic Code Expansion Accessible

    Type
    Blog Post
    Published
    June 15, 2023, 1:15 p.m.
    ...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...

  36. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    Published
    May 20, 2014, 2:06 p.m.
    ...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 ...

  37. Hot Plasmids - October 2022

    Type
    Blog Post
    Published
    Oct. 6, 2022, 1:15 p.m.
    ...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...

  38. Antibodies 101: Buffers, Storage, and Conjugates

    Type
    Blog Post
    Published
    Feb. 1, 2022, 2:15 p.m.
    ...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...

  39. Luminescent Imaging with Nano-lanterns

    Type
    Blog Post
    Published
    May 25, 2017, 2:30 p.m.
    ... 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...

  40. Antibodies 101: Conjugation

    Type
    Blog Post
    Published
    March 26, 2024, 1:15 p.m.
    ...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...

  41. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    Published
    March 6, 2018, 1:49 p.m.
    ...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...

  42. Science as Art

    Type
    Blog Post
    Published
    March 2, 2015, 3:13 p.m.
    ...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 ...

  43. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...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...

  44. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    Published
    Feb. 8, 2018, 12:17 p.m.
    ...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...

  45. Fluorescent Proteins 101: Green Fluorescent Protein (GFP)

    Type
    Blog Post
    Published
    May 15, 2014, 3:33 p.m.
    ...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...

  46. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    Published
    Nov. 16, 2021, 2:15 p.m.
    ... that targets the primary is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between...exist and you can begin to see why generating fluorophore-conjugated versions for each primary antibody...IgGs and then conjugate them with an array of fluorophores. It is common for labs to keep a few trusted... secondary antibodies conjugated to distinct fluorophores to visualize multiple targets at once. An advantage...to ensure that you are using secondaries with fluorophores whose excitation/emission spectra do not overlap....  Fixation Good fixation maintains sample morphology with minimal impact to the target epitope. There...to the first antibody and is conjugated to a fluorophore. You may need to optimize the concentration and...

  47. Plasmids 101: Biotinylation

    Type
    Blog Post
    Published
    Nov. 15, 2018, 1:50 p.m.
    ...binding. Photobiotinylation Photobiotin can be used to non-selectively label DNA and RNA. Photobiotin acetate...papers and webpages are listed below: EMSA (Electrophoretic Mobility Shift Assays) ELISA (Enzyme-Linked...consists of a biotin molecule attached to a photoreactive arylazide group. Under strong visible light,...nearby nucleic acids (McInnes et al., 1990). Photobiotinylation is sometimes easier and less expensive than...C., & Symons, R. H. “Preparation and uses of photobiotin.” Methods in Enzymology 184 (1990): 588–600. ...Schwartz, S. A. “Biotinylated probes in the electrophoretic mobility shift assay to examine specific dsDNA...

  48. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    Published
    April 24, 2015, 2:39 p.m.
    ...caused partially by fluorophore photoinstability, as well as the tendency of fluorophores to aggregate in ...permit analysis by immunostaining. Although the fluorophores used are fluorescently distinct, their protein...preventing design of antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained...ideal characteristics (low aggregation, high photostability, and high stability after fixation.) From the...3.0, but with mOrange2, EGFP and mKate2 as the fluorophores; mOrange2 is the default state. In contrast,...cells in which the construct is expressed. The photostability of Brainbow-3.2 is improved due to the addition...

  49. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    Published
    Nov. 3, 2020, 2:15 p.m.
    ...engineering channelrhodopsins to have improved photocurrent properties: The sequence and structures that...channelrhodopsin, as well as predict the channelrhodopsin’s photocurrent strength, wavelength sensitivity, and off-kinetics...predicted channelrhodopsin sequences would have photocurrent properties best suited for less-invasive optogenetics...All thirty were functional and their measured photocurrent amplitude, wavelength sensitivity, and off-kinetics...neurons expressing ChRgers produced stronger photocurrents at low light intensity than existing channelrhodopsins...response. This result showed that ChRger2 stronger photocurrent properties make it better suited for systemic...ChRger2’s improved light sensitivity and higher-photocurrent properties when paired with PHP.eB’s systemic...

  50. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

    Type
    Blog Post
    Published
    May 15, 2018, 12:43 p.m.
    ... fused to the phosphopeptide binding domain FHA. The FHA1 domain binds to phosphorylated PKA substrate...fluorescence intensity due to interactions with photoactivatable fluorescent protein Dronpa. The authors tracked...detect PKA activity can be generalized to measure phosphatase activity, second messenger activity, and protein-protein...peak of 519 nm. These proteins have improved photophysical properties and can be used in quenching, FRET...
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