...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...

... 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...

...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...

...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...

...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...

... 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...

...-fold more photostable and as bright to mVenus, the parent YFP, and is the most photostable YFP to date...improved activity as compared to other recombinase/photodimer pairs. Created with BioRender.com In their... publication, Mary Dunlop’s lab generated a photosensitive Cre, Opto-Cre-Vvd, by splitting and joining...joining the protein regions with Vivid (Vvd) photodimers. Upon the application of blue light, Vvd dimerizes....ncbi.nlm.nih.gov/32675376/ mGold, the most photostable yellow fluorescent protein to date By Justin ...contribute to experiments that require high photostability, such as time-lapse imaging or with high light...

...I’ve answered hundreds of phone calls and thousands of customer service emails in my six years as a senior...now spend far less time answering help emails and phone calls, I feel compelled to share my years of accumulated...customer, can get the most out of your email or phone call. Though my experience is based solely on my...texting, actually writing an e-mail or making a phone call is a big commitment when you’re busy. Your ...might think. The Addgene scientists answering the phone are smart, but they cannot (and should not) answer... our thread or remove a good 5 minutes from our phone call. If your concern is based on data you have... Abcam and NEB have Ph.D.s answering emails and phones). We can’t be experts on every technique or experiment...

...cells lines have similar morphologies, being familiar with these morphologies and doing a quick check ...that any medium or washing buffer, such as PBS (Phosphate Buffered Saline), that comes in contact with the...purpose.Learn about the effects of CO2 on sample pH Cell morphology: Shape and size matter You should have a light...incubator). It also helps to get familiar with the morphology of the cell line you’re working with so that ...passaged. This could change their growth rates and morphology, rendering their continued use inadvisable for...passage number. Cells change their growth rate and morphology at high passage numbers (>30-40 times), depending...

...an opsin (a GPCR) linked to a chromophore and functions as a photoreceptor in the eye. There are two flavors...voltage-gated potassium channels found in Drosophila to phosphatases from sea squirts. This design structurally separates...cell level as they are in vitro. Factors like photobleaching, lack of red-shifted indicators, and brightness...significantly brighter and available with red-shifted fluorophores (find FRET capable GEVIs here!). The only issue...issue with these GEVIs? They perform poorly in two-photon conditions, which are necessary conditions for ...indicator. To give an example, a genetically encoded phosphatase (voltage sensing) hydrolyzes a precursor dye ...

...all of the fluorophores as it passes through the specimen, which often leads to photobleaching.  Furthermore...focus light. Therefore, there is much less photo-bleaching or photo-damage than in conventional laser scanning...order to improve axial resolution and reduce phototoxicity for imaging live specimens, they combined light...light scattering and absorption.  Finally, the photomultipliers used to detect light in confocal microscopes...obstacles (e.g. air bubbles or high concentration of fluorophores) cast shadows along the illumination path. The...

...identification of plasmid multimers. Capillary Gel Electrophoresis (CGE) analysis Another technique that can detect... detect multimers is capillary gel electrophoresis (CGE), which has been used in plasmid vaccine and gene...Plasmid Topology Monitoring by Capillary Gel Electrophoresis. Curr Mol Med. 2020;20(10):798-805. doi: 10.2174... He Y, Lacher NA, Ruesch MN. Capillary gel electrophoresis with laser-induced fluorescence of plasmid ...plasmid DNA in untreated capillary. Electrophoresis. 2010 Jul;31(14):2436-41. doi: 10.1002/elps.201000061. PMID...topoisomers, knots, and catenanes by agarose gel electrophoresis. Methods Mol Biol. 2009;582:11-25. doi: 10.1007...topology analysis of plasmid DNA by capillary gel electrophoresis. BioProcess Int. 2006 Sep;4(8):38-40. Summers...

... Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first step of a western. ...s post-translational modifications such as phosphorylation, acetylation, methylation and ubiquitination...ubiquitination. For example, to test if a protein is phosphorylated following a specific experimental condition you....2015.04.012 Burnette WN (1981) “Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide....76.7.3116 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels...

... construct for every application. Plus, the photophysical properties of chemical dyes provide many advantages...strategies. The green structure represents a fluorophore or other useful ligand, gray structure is the...non-natural amino acids to directly incorporate a fluorophore in your protein or to incorporate an alkene to...Organic dyes are typically both brighter and more photostable than fluorescent proteins (often up to 10-fold...years; improving the labeling kinetics or the fluorophore’s microenvironment to enhance brightness, stability...Better Dyeing Through Chemistry & Small Molecule Fluorophores. Addgene Blog. https://blog.addgene.org/better-dyeing-through-chemistry-small-molecule-fluorophores...better-dyeing-through-chemistry-small-molecule-fluorophores Los, G. V., Encell, L. P., McDougall, M. G.,...

...sensor’s photophysical properties such as kinetics, ∆F/F0, SNR, dynamic range, photostability, quantum...signals upon sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation...membrane trafficking and localization. Improved photophysical properties The improved variants of iGluSnFR3...pMinDisplay vector. GPI contains a C-terminal glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane...

...Xin Zhou greatly expanded that list by using photoswitchable fluorescent proteins to create enzymes that...turned the enzymes back on. Xin termed these photoswitchable enzymes Fluorescent Light-inducible Proteins...various designs, Xin has been able to develop photoswitchable Intersectin, Cdc42, Raf1, Mek1, Mek2, Cdk5,... You can learn more about Xin’s work with photoswitchable components of the ERK signaling pathway in ... the production of HMOs. Current HPLC-MS and fluorophore-based technologies are too time consuming in ...Resources on the Addgene Blog Learn More about Photosactivatable Fluorescent Proteins Use Light to Control ...