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...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...

...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...

... 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...

...confocal and widefield imaging, two-photon microscopy, and fiber photometry. Plus, both designs offer promising...had better brightness, signal-to-noise ratio, photostability, and signal response upon Ca2+ binding. Most...importantly, it doesn’t display the problematic photoswitching upon blue illumination that plagues other red...mApple, which means it likely has the blue-light photoswitching issue). Although similar in brightness to jRGECO1a...calcium sensor with exceptional brightness, photostability, and multiplexing capabilities. bioRxiv 2024.12.16.628673...incubate at room temperature, and image with smartphones. The Qi lab also developed an algorithm called...

...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...