We narrowed to 321 results for: POR C

...variables. Twitch is a FRET-based calcium reporter derived from troponin C. It is engineered with a reduced number...calcium binding proteins (e.g., calmodulin or troponin C). Their fluorescent readouts are used to monitor calcium...Calcium is arguably one of the most important metal ions in biology (sodium and potassium are up there..., and an allosteric modulator. Because of its importance, scientists have developed calcium sensors to...GCaMP6 family of sensors are commonly used calcium reporters with different kinetic parameters. There are three...also available. Detection methods Many calcium reporters use a single fluorescent protein (FP), requiring...for in vivo studies) There are several calcium reporters specially designed for the aims and requirements...

...connected by a 9 amino acid linker that’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of...are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott, M., ...spectra. DsRed is the permanent fluorescent tag portion of Rosella: it will emit red fluorescence regardless...mechanisms behind autophagy by tracking what’s being transported to the yeast vacuole (i.e. cytosol, mitochondria...2008). Rosella: A fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in...using a single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030...

...from an additional 5–10 minutes of incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh ...stripping solution and agitate for 30–45 minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh ...directly to buffer without a re-wetting step. Important: do not dry your membrane with antibodies on it...the ones shared by Bio-Techne, ThermoFisher, or Millipore Sigma. We’ll share the basic composition of these...Alternatively, companies including ThermoFisher, Millipore Sigma, and Azure Biosystems offer pre-made stripping...your western blot several times — some researchers report up to ten cycles! Just remember that a bit of sample...

...blog post for more details. Figure 2: Dense (c), intermediate (d), and sparse (e) labeling of the...remove a stop cassette upstream of an effector/reporter gene. In cells where the stop cassette is removed...https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article contributed... it is an attractive drug target. Calcineurin reporters allow scientists to measure calcineurin dynamics... target calcineurin. Current calcineurin gene reporters are based on the bacterial βgal assay, and are...Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized ...panels) equipped with destabilized GFP calcineurin reporter (blue) and stained with PI (pink) to indicate ...

... its preferred target integration site: TNA(C/G)TTATAA(G/C)TNA. Tol2 can deliver 10-11 kB to mammalian...   References and Resources References Feschotte C, Pritham EJ (2007) DNA Transposons and the Evolution...piggyBac plasmids    Tol2  Tol2 was the first reported active DNA transposon in vertebrates. It was discovered...mutations. In these screens, the transposons encode reporter genes, mutagenic cassettes, or barcodes. When ...2021). Target site preference and host range are important to consider when selecting a transposon for a ...pronuclei of fertilized eggs, which leads to random incorporation of that sequence into the zygote’s genome, a...unpredictable. Transposons, however, efficiently incorporate into the zygote’s genome after injection into...

...application of blue light, Vvd dimerizes bringing the C-terminal and N-terminal Cre fragments together and...the PAM sequence is 5’-TBN-3’ (where B is G, T, or C). The authors demonstrate that CRISPR-Casɸ is active...in a protein.    Cells expressing an RFP reporter plasmid for recombinase activity were exposed ...interest. However, FACS is unable to spatially and temporally target single cells. Now, Francois St-Pierre’...way to isolate individual cells with unique spatiotemporal phenotypes from large heterogeneous cultures...introduction & resolution of two DSBs in a fluorescent reporter. Find the plasmids here Yongsub Kim’s lab engineered...

...These plasmids enable lentiviral expression of N- or C-terminally FKBP12F36V-tagged fusion chimeras or locus-specific... rapid means of comparing functionality of N- and C-terminal fusions and degradation in a cell line of...transcription factors with important roles in Multiple Myeloma), it is important to ensure lack of degradation... chimeras For each protein-of-interest, it is important to determine which terminus can accommodate the...

...easy to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus, and selection...Griffith, A. L., Szegletes, Z. M., Wen, B., Kraus, C., Miller, N. W., Steger, R. J., Escude Velasco, B....New to the bench? Fragmid depositor John Doench reports even the greenest members of his lab, high school...vectors are available? What you’re building is important — but so is where you’re building it! Table 1 ...

...doi.org/10.1128/AEM.67.8.3440-3444.2001 Fogarty, E. C., Schechter, M. S., Lolans, K., Sheahan, M. L., Veseli....102684 Shareck, J., Choi, Y., Lee, B., & Miguez, C. B. (2004). Cloning Vectors Based on Cryptic Plasmids...plasmids — more on that in a minute! Fun fact! Electroporation is one popular method scientists use to promote...

... was created by adding a 9-residue peptide to the C-terminus of human codon-optimized  cryptochrome 2 ...a fragment of the kainate receptor subunit to the C-terminus of the protein. Unlike the diffuse neuronal...while also capturing information about spatial and temporal organization. The design use to detect PKA activity...

...mutations in the common cytokine receptor gamma chain (c) and is characterized by a lack of immune cell development...1172-1179. PubMed PMID: 28671695. 6. Münch, Robert C., et al. "Off-target-free gene delivery by affinity-purified...basic research and other ongoing clinical trials support the utility of AAV as a broadly applicable gene...

...Most FP fusions for FRET studies are at the N- or C-terminus of your target protein, but even these modifications...can insert your protein of interest, resulting in a C-terminal fusion of a tetracysteine tag. In addition...for observed FRET efficiencies between 80% and 10%. C Cy3NTA and Cy5NTA bind His tags similarly but with...nonspecifically soak up your fluorophore package. One important concept in FRET experimental design is that the...donor first where labeling specificity is most important. Donor labeling and the need for specificity Donor...protein molecules contain the donor and, more importantly there is no fluorescence background resulting...in the ribosome, resulting in site-specific incorporation of the attached fluorophore. However, since ...

...in mice. Scientific Reports 9: https://doi.org/10.1038/s41598-019-41088-2 Magnus C, Lee P, Bonaventura...10.1021/cn500325v Goutaudier, R., Coizet, V., Carcenac, C., & Carnicella, S. (2019). DREADDs: The Power of the...Targeted and specific delivery becomes especially important for studies in neuroscience where specific tissues...neuron need to be targeted. Targeting is also important in the development of therapies when expression...Picking Ligands Picking the right ligand is also important for building a chemogenetic system. Your experiment...cellular response you aim to manipulate. It is important to consider that the same receptor can elicit ...drug that only binds to one receptor, so it is important to fully characterize the compound, or compounds...

... CRISPR plasmids deposited with Addgene by year. (C) Cumulative number of laboratories that have deposited...smoothed using locally estimated scatterplot smoothing. (C) Number of requests per year for pooled libraries,...plasmids/year from 2017 to 2023. This growth is supported by new labs joining the Addgene CRISPR community...year of deposit. Outlines show a representative proportion of CRISPR plasmids requested, categorized by ...plasmids deposited by country; square size is proportional to number of plasmids deposited by each country...plasmids requested by country; square size is proportional to the number of plasmids requested by each ...Barrangou for his authorial contributions to, and support for, this and previous Addgene CRISPR publications...

....2307/1542146 Lazarus, J. E., Warr, A. R., Kuehl, C. J., Giorgio, R. T., Davis, B. M., & Waldor, M. K..... D., Jamal, S., Wistrand-Yuen, P., … Forster, A. C. (2018). Engineering a palette of eukaryotic chromoproteins...Characterization of the Pink and Blue Pigments of Pocilloporid and Acroporid Corals. The Biological Bulletin 189:288...chromoproteins and fluorescent proteins, it’s important to note that despite their distinction by name...interactions with cytosolic proteins. This step is important as non-specific binding to proteins can inhibit...improved expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604...

...these methods a try.  References Højland Knudsen C, Ásgrímsdóttir ES, Rahimi K, Gill KP, Frandsen S, ... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide ...Recently scientists have created CRISPR fluorescent reporter assays to quantify targeted Cas9 cleavage, and...and non-homologous end joining (NHEJ). In these reporters, editing typically results in a change in the ...this blog we will focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover...

...Maddalo D, Manchado E, Concepcion CP, Bonetti C, Vidigal JA, Han Y-C, Ogrodowski P, Crippa A, Rekhtman N, de...Scientific Reports paper introducing the delivery method in 2014, Manuel Goncalves lab report that AdV-...the likelihood that donor DNA will interact with sporadic double-stranded chromosomal DNA breaks “that always...

...Analyze a Restriction Digest References 1. Henrici, R. C., Pecen, T. J., Johnston, J. L., & Tan, S. (2017)....Generating DNA Molecular Weight Markers. Scientific Reports,7(1). doi:10.1038/s41598-017-02693-1. PubMed PMID...

...References and resources References Chadeuf G, Ciron C, Moullier P, Salvetti A (2005) Evidence for Encapsidation...Lindenbaum P, Martin-Fontaine M, Broucque F, Robin C, Hebben M, Merten O-W, Blouin V, François A, Redon...production process? These questions are especially important when producing multiple vectors in parallel because...

...temperatures (up to 95 °C) and is active over a range of elevated temperatures (37–75 °C). T4 DNA ligase ...PMC4737175. 22. Liu, Pingfang, Artur Burdzy, and Lawrence C. Sowers. "DNA ligases ensure fidelity by interrogating...commercially available (see below). Conclusion It is important to note that thermostable, high fidelity, nick-selective...

...Discovers America. New York./  Callahan, K., Peterson, C., et al. 2022. External collaboration results in student...Evolution and Development. 24(5):127-130. Kimball, C., Powers, K., Dustin, J., Poirier, V., Pellettieri... outcomes and the opportunity to make new discoveries. Provost notes the importance of acknowledging student...history. Interestingly, he also adds to the disproportionately large number of honorees to have trained ... and he wanted to provide the same kinds of opportunities for his students. But he also found that his...Bringing his research into the classroom. I had the opportunity to talk with Provost recently about his development...Undergraduate Research Experience (CURE). Having incorporated a discovery-based lab on planarian regeneration...

...conformation capture (3C) based methods, such as 5C or Hi-C (Han et al., 2018). Think of 3C methods as taking ...other 3D chromatin methods such as ChIA-PET or Hi-C, by increasing both the number of unique chromatin...but enhancers and chromatin looping play important supporting roles. Enhancers are cis regulatory DNA ...networks’ of 157 super enhancers.  3. Measuring temporal changes in chromatin interactions A third application...application for CAPTURE 2.0 is detection of temporal changes in chromatin conformation, like those that ...interactions. The chromatin story told by CAPTURE 2.0 is important, but it’s not the only plot line in gene regulation...Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated...

... epitope such as c-Myc, GFP or V5 can be used to tag the protein of interest on the C- or N-terminus. ...Their porous surface provides a large area for binding, but antibodies conjugated within the pores are ...used for IP. These proteins bind only to the Fc portion of the immunoglobulin, leaving the antigen binding...or a magnet (magnetic beads), providing a solid support while unbound proteins are washed away.  While ...to be adjusted depending on the sample. It is important to remember that sample lysates will also contain...

...predictable kinetics over 14 hours of expression in C. elegans embryos. However, this initial timer was ...timer could be used in vitro, in mammalian cells, in C. elegans, and in Xenopus embryos. Of course, timers...have been around since 2000 when Terskikh et al. reported the production of dsRed E5. This timer predictably... your experimental setup less cumbersome. 3. Portability across multiple systems Terskikh et al. showed...fluorescent timers that change color from blue to red report on cellular trafficking." Nature chemical biology...Chronology of islet differentiation revealed by temporal cell labeling." Diabetes 58.8 (2009): 1863-1868...

...calculate the viral titer: GC/mL = {[(R*C)(1000/V)]*D} R = Reaction volume C = Copies/uL V = Volume of virus ...by ddPCR begins with diluting the virus. It is important to note that the dynamic range of the ddPCR is...PCR plate. Maintaining a high droplet count is important for calculating the concentration of your sample...separation (Witte et al., 2016). This is particularly important if your amplicon is longer than 150 base pairs...

...overhangs Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C (not required by all orthologs) Specific RNA cleavage...spacer sequence and consists of a single A, U, or C base pair. In bacteria, once Cas13a has recognized...of interest and an inactivated fluorescent RNA reporter. If the target sequence is present in the pool...activity of Cas13a becomes activated and the RNA reporter will be cleaved resulting in activation of the...in the field and as always, we appreciate the opportunity to share these invaluable reagents with the scientific...

...https://doi.org/10.1096/fj.01-0995fje. Tan, E., Chin, C. S. H., Lim, Z. F. S., & Ng, S. K. (2021). HEK293 ...doi.org/10.3389/fbioe.2021.796991. Urabe, M., Ding, C., & Kotin, R.M. (2002). Insect cells as a factory ...of production, saving about a week of work. An important note is that this is only possible because baculoviruses... will come down to which differences are most important to you — virus type, cost, time, or reagents and...

...construct. Reactions are often cycled between 37°C and 16°C to provide optimal reaction temps for both the... Golden Gate assemblies feature an inverse proportionality between the complexity of the assembly (number...The future of assembly DNA assembly methods are important tools for many areas of science, and researchers...

...our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq DNA...commonly think of DNA as a linear polymer of A,T,G, and C nucleotides arranged in a double helix. People are...genome. But not all of those 13,000 regions are important for maintaining ESCs. So which of these 13,000...next-generation sequencing to measure the relative proportion of guide RNAs (gRNAs) across the population. ...

...tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses of the affinity ... tags, a His tag can be fused to either the N- or C-terminus of a protein. Unlike other epitope tags –... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the... are available, but scientists would need to incorporate their recognition sites into their protein tag...

...into an attL-containing entry vector.   Method C: Restriction cloning of a restriction enzyme fragment...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning the ...coding sequence. If you choose this strategy, it’s important to include the proper protein expression elements...Clone When making the expression clone, it is important to choose the destination vector that best fits...

...create an N- or C-terminal fusion based on your experimental needs. Equally important, before cloning ... retrieval sequence only functions at the extreme C-terminus [3]. You must therefore determine your cloning...potentially affect function [5]. We and others have reported that FP cysteine residues form inappropriate disulfide...

...fluorescent proteins (GFP and YFP) and protein tags (c-MYC and HA). We have also created the Monoclonal ...repositories, such as Addgene and the DSHB, play an important role in scientific exchange. In addition to ensuring...provide standardized quality-control and technical support to the community. Thanks to Our Guest Blogger! ...

... a AAV8 expressing eGFP under the human ubiquitin C promoter. Anatomical abbreviations: Gr= Gracile tract...Deisseroth. (2014) Nat Protoc., 9, 1682-1697. Pubmed. C. Soderblom, D.-H. Lee, A. Dawood, M. Carballosa, et...addition, the use of viruses allows much better spatiotemporal control over labeling methods as compared to...

...similar nuclease folds found in other proteins. The C-terminal domain (CTD) of the protein also belongs ...https://doi.org/10.1038/s41586-022-04470-1 Cofsky, J. C., Soczek, K. M., Knott, G. J., Nogales, E., & Doudna...Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C., & Doudna, J. A. (2014). DNA interrogation by the...it is limited to only 20 bases, or why it’s so important to choose a target sequence with a nearby protospacer-adjacent... lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is so flexible..., but they improve cutting efficiency and are important for robust activity in cells (Jiang & Doudna, ... guide RNA stabilizing the DNA-binding protein portion of the CTD, ready to check for PAM sequencines....

...References and resources Milling, L. E., Markson, S. C., Tjokrosurjo, Q., Derosia, N. M., Streeter, I. S....screens have been used to interrogate T cells, an important immune cell type, in more complex and physiological...

...reactions, where a fraction of one of the four bases (A, C, T, or G) is supplied as a dideoxyribonucleoside triphosphate...regions of interest, or complex sequences such as G-C rich areas, secondary structures, or repetitive regions... technology, but Oxford Nanopore is one of the most popular options. Nanopore sequencing deviates from...pulled through the pore via motor proteins. When each nucleotide passes through the nanopore, the electrical...3: Overview of Nanopore sequencing technology. Created with BioRender.com. Nanopore can provide longer...sequencing methods (Figure 3). It uses (aptly named) “nanopores” that are attached to the flow cell membrane and...-read sequencing. At Addgene, we routinely use Nanopore technology to sequence our packaged viral DNA ...

...these methods a try.  References:  Branon, Tess C., et al. "Efficient proximity labeling in living cells...1837. PubMed PMID: 30676735. Kim, D. I., Jensen, S. C., Noble, K. A., KC, B., Roux, K. H., Motamedchaboki...the mitochondrial calcium uniporter (Martell et al., 2012). It is important to note that the biotin-phenol... of exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo... where two proteins are fused to complementary reporter proteins that can only assemble when in close ... ascorbate peroxidase as a genetically encoded reporter for electron microscopy." Nature biotechnology...proteomics approach to monitor the composition of spatiotemporally defined protein complexes." Nature communications...

...specificity of GFP expression for the indicated ssAAV5s. C) Quantifications showing high microglia transduction... microglia-targeting AAV plasmids here! Serrano, C., et al. (2023). Simple and Highly Specific Targeting...2024). They developed a new prime editor that incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7...high microglia-specific gene expression when incorporated in an AAV vector (Figure 5). Efficiency and ...including in human retinal organoids and live mice. Importantly, REVeRT avoids the introduction of extraneous...associated with viral vector-based therapies. REVeRT supports advanced gene expression regulation, enhancing...

...due to poor folding and thermal instability at 37°C (Ouellet et al., 2016) and requires a tRNA scaffold...imaging single molecules and for live-cell imaging in C. elegans. Other groups have used Mango to study RNA-protein...split aptamer is composed of a “Top” and “Bottom” portion, each fused to a separate piece of RNA. Fluorescence...photostable, and were presented in the study to support the uniqueness of the photostability of the Corn-DFHO...growth as RNA biology, we are looking forward to reports on the next additions to aptamer soup! References...

...frozen glycerol stocks. It is very important that these stay frozen at -80 °C, since multiple rounds of freezing...; CDSns, coding sequence with no stop codon; TAG, C-terminal tag; TER, terminator. Created with BioRender.com... perfect Our first tip, and arguably the most important, is to take some time to sit down, plan your cloning...

...into single-use aliquots, and stored at -80°C. Some reports suggest rapidly freezing virus in a dry ice... for less than a day, lentivirus can be kept at 4°C. For long-term storage, viral preps should be divided...protein attaches to cell surface receptors, other important binding events take place during the course of...

...conditions. Many recombinant proteins express better at 30°C or room-temperature, which is accomplished by growing...growing your culture to the desired density at 37°C and reducing the temperature or moving it to a bench-top...added to the growth medium. Induction timing is important, as you typically want to make sure your cells... optimal because of the high cell-density they support; however, minimal media supplemented with M9 salts...

...entire gene? Perhaps you are more interested in the c-terminus and would like to target that region. Or ... can be cloned directionally into the BbsI site. (c) The pCAG-EGxxFP target plasmid was co-transfected...expressing Cas9 and single guided RNA." Scientific reports 3 (2013). PubMed PMID: 24284873. PubMed Central...

...Zhi Ying Chen, Robert C. Robbins, Mark A. Kay, Michael T. Longaker, and Joseph C. Wu. "A nonviral minicircle...a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs." Plos One 11, no...of Different Methods for Generating iPSCs. One important note: in this table, reprogramming efficiencies...high is based on the reprogramming efficiencies reported in González et al. A percentage is not listed ... be used to package reprogramming factors. An important milestone of reprogramming is the silencing of...cells for the presence of transgenes is still an important control. Additionally, Sendai virus doesn’t rely...comprised of OCT4, SOX2, LIN28, NANOG, and a GFP reporter. Minicircles are removed from cells by dilution...

...’s most common diseases. References Zhang Y, Yin C, Zhang T, Li F, Yang W, Kaminski R, Fagan PR, Putatunda...Gendron P, Zhu W, Guo F, Cen S, Wainberg MA, Liang C. CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 ...infection, including both common infections and opportunistic infections not usually seen in healthy individuals... genome had been excised, leaving only a small portion of the LTRs joined together. Clonal Cas9/gRNA-expressing...populations were also immune to HIV-1 reinfection. Importantly, no off-target effects were detected. Kaminski...patients. Although this is an exciting finding, it’s important to note that these HIV-1 patients were ART-naive...Cas9. While these results are discouraging, it’s important to note that these experiments were done in cell...

...Sato T, van Es JH, van den Born M, Kroon-Veenboer C, Barker N, Klein AM, van Rheenen J, Simons BD, Clevers...melanogaster. Hadjieconomou D, Rotkopf S, Alexandre C, Bell DM, Dickson BJ, Salecker I. Nature Methods. ...the 1980s, is one of the most important ways to spatially and temporally control gene expression, especially...ten colors (see table to the right); in various reports, Sanes and Lichtman have observed from 90-160 distinct...undergone recombination. This default state was disproportionately expressed by a majority of neurons, limiting... 8 colors. The use of AAV provides spatial and temporal control without the need for germline modification...refinements of the Brainbow technique will provide important insights into the complicated physical organization...

...expressed GFP in exogenous organisms (E. coli, and later C. elegans) for the first time. After Chalfie’s work...responsible for encoding GFP, and, in 1992, he reported its sequence. Soon after that, in 1994, Prasher...he lived! His hunch paid off, and he was able to report new fluorophores like DsRed from Discosoma sp. ...provided by the studies in Tsien lab, covered a portion of the color spectrum that GFP and its derivatives...reach. Nobel Prize for fluorescent proteins The importance of fluorescent proteins was recognized in 2008...

...involve a huge group getting from point A to B to C to D, etc. 7. Don’t let a tiny [or non-existent] ...that company culture and enjoying your job were important parts of team buidling - they relieved some of...

...solubility (Monnier et al., 2013). The linker keeps the C-terminus of one variable domain and the N-terminus...10.3390/antib2020193  Khantasup K, Chantima W, Sangma C, Poomputsa K, Dharakul T (2015) Design and Generation...cells to make CAR T cells. The extracellular scFv portion of the receptors recognizes an antigen of interest...interest while the intracellular T cell receptor portion of the CAR facilitates signal transduction and release...