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We narrowed to 727 results for: cat.1

Showing: 121 - 150 of 727 results

  1. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

    Type
    Blog Post
    ...sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation and ...hSynap: Human Synapsin-1 promoter. Good for neuronal expression. hSynap-FLEX: Synapsin-1 promoter, FLEXed for... v857. Image adapted from Aggarwal et al., Figure 1   Increased versatility through two different...expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent expression GFAP: Glial fibrillary...Glutamate plays a variety of roles in synaptic communication and can trigger other forms of neuronal signaling...SF-Venus-iGluSnFR). iGluSnFR3: Improved Glutamate Indicators for Synaptic Imaging However, the first two generations... 30377363 Aggarwal, Abhi, et al. . “Glutamate indicators with improved activation kinetics and localiation...

  2. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...excision repair mediated by uracil glycosylase (Figure 1). The presence of a uracil glycosylase inhibitor (...generation of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation... A-T base pair is converted to a G-C pair (Figure 1). As the cellular inosine excision repair is not as...Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful base editors in rice, tomato...deaminase can edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong et al., 2018...which recognize PAM sequences other than NGG (Table-1). Type Nickase Cas9 (D10A) Deaminase PAM requirements...locus/loci, choose a base editor variant from Table 1 depending on the PAM sequence. Avoid targeting a ‘...

  3. An Integrin Antibody Toolkit from IPI

    Type
    Blog Post
    ...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.2] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...-Integrin alpha 5 beta 1 [IPI-ITGA5/ITGB1.4] Integrin alpha-5,Integrin beta-1 Human, Mouse IgG1 Rabbit...subtype-specific β1 integrin mechanics. Nature Communications, 13(1), 7471. https://doi.org/10.1038/s41467-022...specific integrins in cellular processes.   Figure 1: Integrins can shape into three conformations: (A)...immunoprecipitation and immunohistochemistry. Table 1: IPI antibodies  Antibody Target Proteins Reactivity...Human IgG1 Rabbit Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG1 Rabbit Anti-Integrin...Human IgG2a Mouse Anti-Integrin alpha M [IPI-LM2/1] Integrin alpha-M Human IgG2a Mouse Anti-Integrin...

  4. Hot Plasmids February 2024

    Type
    Blog Post
    ...Biotechnology, 10.1038/s41587-023-01756-1. https://doi.org/10.1038/s41587-023-01756-1. PMID: 37095348. Breaking...molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038/s41592-023...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023...incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and...expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under...StayGold fluorescent protein. Nature Biotechnology, 1–4. https://doi.org/10.1038/s41587-023-02018-w. PMID..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric...

  5. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    ...to the most colonies after transformation. When a 1:1 vector:insert ratio is used in the DpnI digestion... get tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and...Connecticut interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis...PCR cloning method." BMC biotechnology 11.1 (2011): 1. PubMed PMID: 21992524. PubMed Central PMCID: PMC3207894...Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - Ligation + - Transformation...vector is located in the 3’ end of the multi-cloning site, and the reverse primer is located in the 5’...Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without sequence alteration...

  6. Addgene is Expanding Our Viral Vector Service!

    Type
    Blog Post
    ...  Request review: 1–2 days Prep and QC completed: 6–8 weeks Average shipping time: 1–2 days (USA); 3–4...choice of five different serotypes.   Figure 1: The process of requesting and ordering a Packaged...request in three different quantities: 10x 100 µL (1 mL), 25x 100 µL (2.5 mL), or 50x 100 µL (5 mL).  Once...with our production process. This process will take 1–2 days, and we anticipate approving the vast majority...request is approved, you will receive an email notification that your request is ready to order. An “Add...subscribe to the Addgene blog for updates and educational resources.  We hope you’re as excited about this...

  7. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ...created by making some or all of the following modifications: 1) increased concentration of Taq polymerase...introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al). It uses a uracil labeled wild-...primers to introduce all desired codon changes (step 1 in the image above). A second universal primer is ...strand is nicked by the BbvCI variant not used in step 1. Then this strand is degraded with ExoIII and ExoI...number of positive clones. See Supplementary Note 1 in Wrenbeck et al for details on how nicking was optimized... strand of DNA degraded in the first digest (step 1 in above diagram). Remember that the BbvCI enzymes...time no, but there are 2 instances where it does. 1) When the backbone vector has multiple BbvCI sites...

  8. Cancer, Inflammation and Immunity - Harnessing the Body’s Defenses to Fight Cancer

    Type
    Blog Post
    ...aggregation, and potentiates the effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system...fasting has a positive effect on cancer treatment (Ref 1). As described in the presentation, starvation or ...of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment in turn may represent...  (Chimeric Antigen Receptors) CAR T cells and PD-1 modulators. An exciting development in immunotherapy...free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...Immunosurveillance and Defense With increasingly sophisticated visualization techniques it is possible to follow...

  9. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team will communicate with your ...scientist, you will have the most involvement with Step 1, and, while data entry can be a chore, we’ve made ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit Online...spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you to choose...and are exceptions to our standard workflow.  Table 1: Guidelines for the data fields in the deposit spreadsheet...species can be indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid...

  10. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ... strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different DNA strand...Let’s break down the steps needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers ...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...enzyme and Taq, with Taq used in an excess ratio of 10:1. Gel purify your PCR product and incubate it with ...you to see colonies within 8 hours. References: 1. Shuman S. "Recombination mediated by vaccinia virus...insert comes from using Taq polymerase for the amplification step since Taq polymerase leaves a single deoxyadenosine...rejoining supercoiled DNA ends to facilitate replication. The TOPO technique specifically uses Vaccinia...

  11. Adenovirus Guide

    Type
    Guide
    ...to an antigen. AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid. Adenovirus...icosahedral capsids and build new virions. Figure 1: Wild-type adenovirus and its genome. Genes over or... This construct is then transformed into AdEasier-1 cells (Addgene #16399) , which are BJ5183 E. coli ... viruses. The adenoviral backbone plasmid pAdEasy-1 (Addgene #16400) is suitable for most purposes. For...production system. Figure is based on the use of pAdEasy-1. Created with BioRender.com. AdMax™ AdMax™ is another...landscape . Signal Transduction and Targeted Therapy, 6 (1), 53. https://doi.org/10.1038/s41392-021-00487-6 (...as a Zika virus vaccine . Scientific Reports, 10 (1), 3613. https://doi.org/10.1038/s41598-020-60238-5...

  12. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    ...near-perfect physical-to-infectious particle ratio of 1:1 (Zeltner et al., 2010). However, for recombinant ...preparations are genome-containing (Figure 1).    Figure 1: Electron micrograph of an Addgene AAV...reported a physical-to-infectious particle ratio of 50:1 (Zeltner et al., 2010). The specific infectivity of... lot (if we still have it available). References 1. Fagone, Paolo, et al. "Systemic errors in quantitative... Human Gene Therapy, Part B: Methods 23.1 (2011): 1-7. PubMed PMID: 22428975. PubMed Central PMCID: PMC3640491...the other to guess a category by listing off things that fall into that category? Okay, let’s play. I’...’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot ...

  13. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...marker. Looking at Table 1, we can recognize a couple of fluorophores from Figure 1. For example, the chart...efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining...Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective colors of a few commonly... the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly used fluorophores...Brighter fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly used fluorophores...blue laser excites at a wavelength of 488 nm (Table 1), which is near a local maximum of PE's excitation...Knowing that the red laser excites at 640 nm (Table 1), while comparing the two fluorophore's excitation...

  14. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    ... handed out at the event). Program 0 - 1:24 - Intro 1:24 - 29:31 - Reproducibility Overview - Jeffrey... 49:45 0 1:18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2...reproducibility As Jeffrey Flier points out in his talk (1:24 - 29:31), there are a variety of ways of defining...enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments...the Institute for Protein Innovation (IPI) (49:45 - 1:18:45), there are also initiatives to create more ...found. Consider the hypothetical situation in Figure 1 where many scientists are performing similar experiments...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...

  15. 5 Uncommon Ways to Gain Career Skills During an Uncommon Time

    Type
    Blog Post
    ...need a job. Start now. Informational interviews are 1:1 discussions with someone who has interesting experiences...conversation builds a relationship for the future and the 1:1 format is good for everyone including those that ...out-of-the-box ideas that might jump start your learning. 1. Create a peer mentoring group We don’t all get the...be a time when you need to speak publicly to communicate your work. One of my 10 tips for giving a great...; Graduate Student Funding Opportunities Open Education Database: 100 Places to Find Funding For Your ...

  16. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    ...Jacqueline N Crawley  [et al]. 2012;Chapter 1:Unit 1 20 1-18. doi: 10.1002/0471142301.ns0120s59. PMID:...mutually exclusive) commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. ...can be broken down broadly into three main steps: 1) Selection and expression of your opsin of choice ...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation of..., Jacqueline N Crawley  [et al]. 2013;4(435):4 35 1-4  20. doi: 10.1002/0471142301.ns0435s65. PMID: 25429312...

  17. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    ...deletions, point mutations PCR Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes...methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-mutagenesis... the steps of gel purification and in vitro recombination. Across all modifications described in Trehan...mutations, large deletions/insertions require complicated, often costly in vitro assembly methods. A new...recombination machinery, which facilitates easy genetic modification by homologous recombination. In most recombineering...

  18. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    ...holds true in genome-wide screens.   Figure 1: Summary of the advantages of the new approach to ... in both screens, with the growth screen having a 1% false discovery rate and identifying >88% of previously...in Morgens et al. contains three types of guides: 1) targeting guides (blue), which target all protein-coding...off-target sites was profiled. Guides with exact or 1-bp mismatch off-targets had greater toxicity than ...characteristics that influence gRNA off-target activity: 1) mismatches closer to the PAM are less tolerated than.... When comparing the toxicity of guides that have 1-bp mismatch off-target sites, short guides had reduced...Knockout Library are available from Addgene! References 1. Morgens, David W., et al. "Genome-scale measurement...

  19. Plasmids 101: Control Plasmids

    Type
    Blog Post
    ... and investigation. References: 1. The problem of pseudoreplication in neuroscientific studies: is it...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...control and is consistent with the result in Figure 1 could look like this: Figure 2: Expression of Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...two types of replicate controls: technical and biological. In general, technical replicates can be thought...

  20. Celebrating Outstanding CRISPR/Cas9 Achievements at the Dr. Paul Janssen Award Dinner

    Type
    Blog Post
    ...the result was a simpler 1 protein - 1 RNA system.  Dr. Charpentier advocated for the crucial role of ...minutes, which was fun to watch. A simpler 1 protein - 1 RNA system Drs. Dounda and Charpentier met at...adaptive bacterial immunity and its potential applications in rewriting the genome. Their award acceptance... same Polish sub-dialect making their Skype communications extra fun) who shared first authorship on their... area will accelerate the launch of clinical applications. During the panel discussion, Dr. Mello took...for billions of years (…) Bacteria are really sophisticated”.     New to CRISPR/Cas9 Technology? Learn ...

  21. Antibodies 101: Producing Recombinant Antibodies

    Type
    Blog Post
    ...materials have been washed away (Figure 1).      Figure 1: The steps of rAb production. Created ...EBNA1 Cells. Methods Mol Biol. 2018;1850:1-16. doi: 10.1007/978-1-4939-8730-6_1. PMID: 30242676. Frenzel...Neuroscience Research. Curr Protoc Neurosci. 2020 Dec;94(1):e106. doi: 10.1002/cpns.106. PMID: 33151027; PMCID...detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub...addgene.org Addgene's Antibody Purification Protocol Addgene's Video Antibody Purification Protocol Addgene's Antibody...cells can perform human-like post-translational modifications that bacterial and most yeast cells cannot. ...production system may depend on what downstream applications you are planning, cost, and the amount of protein...

  22. Negative Can Be Positive: Open AAV Data with Addgene

    Type
    Blog Post
    ...across scientific disciplines. Scientific Data, 8(1), 1–11. https://doi.org/10.1038/s41597-021-00981-0 Wright...occurrence during vector purification and formulation. Molecular Therapy, 12(1), 171–178. https://doi.org...excellent at infecting our target neurons (Figure 1). However, this one set of experiments didn’t provide...yet none of that data was published.    Fig. 1: Testing of three different AAV serotypes (AAV1, 8...Stimulation. Frontiers in Human Neuroscience, 14(December), 1–12. https://doi.org/10.3389/fnhum.2020.578687 Additional...caudal NST. (A-C) Low-magnification images showing viral spread. (D-F)  High-magnification images immuno-stained... right AAV for your system or to successfully replicate published works. Of particular importance to these...

  23. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ...range of titers. We usually dilute our samples 1:6 million to 1:25 million.  After making the dilutions, they...note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies (GC) per reaction. Since...In the image to the right, there is approximately 1 positive copy per microliter in the no template control...elongation time to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation...diagnosis." Emerging microbes & infections 7.1 (2018): 1-9. PubMed PMID: 29691363. PubMed Central PMCID: PMC5915492...take to achieve a clean NTC: Have a dedicated bench with a dedicated set of pipettes for ddPCR set-up. .... Each droplet contains the ingredients for amplification of the target DNA. This partitioning reduces...

  24. CRISPR Antimicrobials

    Type
    Blog Post
    ..., they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik et...from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting...genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) a second lytic phage...the fight against bacterial superbugs. References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such...

  25. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with the exception...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will...to additional protocols and plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "...

  26. SciComm with the Experts at Science in the News Part 1

    Type
    Blog Post
    ...Science Communication Conversation in Science Communication The Purpose of Science Communication #1 Mistake...think about communicating science. Listen to the interview with Science in the News, Part 1 Podcast breakdown...supporters of science communication. To help you think about ways to effectively communicate your science, we...News at Harvard University. Effective science communication is required to promote public support for research... organization that works to promote science communication. Science in the News hosts a podcast, seminars...That’s right, #ActualLivingScientists, eager to communicate their science. In this first half of our two-...Mistake Scientists Make when Communicating with Non-Scientists How to Approach Different Audiences Listen...

  27. Technical Design of a Western Blot

    Type
    Blog Post
    ...Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability...lot to cover, so let's dive in!      Figure 1: The technical decisions to make when designing a ...assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...weight of the proteins you’re interested in. Table 1 lists the most common types of gels for SDS-PAGE running...gel chemistry and/or specialty gel options. Table 1: Types of gels  Gel Type Protein sizes Running... Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...

  28. Getting to Know Expansion Microscopy

    Type
    Blog Post
    ...protocols. A typical ExM protocol (Fig. 1) follows this structure: Step 1: fix (and maybe label) your sample...microscopy. Nat Biotechnol 1–12. https://doi.org/10.1038/s41587-022-01546-1  Mikhaylova M, Cloin BMC, ...just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify ...16:71–74. https://doi.org/10.1038/s41592-018-0238-1  Klimas A, Gallagher BR, Wijesekara P, et al (2023...with an abundance of protocols and examples of applications from across disciplines now available. In this...experiment or evaluating ExM images in a new publication.         Why Expansion Microscopy?  Traditional...microscopes sounds great, right? But there is always a catch - some limitation of the technique that users should...

  29. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR sites...regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions. ...expression of the human KRAS gene in mammalian cells. STEP 1: Generate an Entry Clone There are a few different...could use a vector like pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible pLIX_403. The chosen...the generation of the expression construct in only 1 day, as opposed to 2+ days with traditional restriction... Find Gateway cloning plasmids here! References 1. Chee JY, Chin CF (2015) Gateway Cloning Technology...attP sites of the donor vector. This reaction is catalyzed by the BP Clonase enzyme mix and generates the...

  30. Lentiviral Vector Guide

    Type
    Guide
    ...typically ranges from 8–10 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...been removed or mutated for increased safety. Figure 1: Wild-type lentivirus genome. Created with BioRender.com...genes (for example, vif , vpr , vpu , and nef for HIV-1). The LTRs flank all of these genes, and anything ...transgene and LTRs (chimeric 5’ LTR) Packaging plasmid 1 — contains gag and pol Packaging plasmid 2 — contains...and third-generation systems, please refer to Table 1. Figure 4: Third-generation lentiviral plasmids. Created...heterologous enhancer/promoter such as CMV or RSV Table 1. Summary of key differences between second- and third-generation...More Lentiviral Vector topics Addgene Protocols pLKO.1 - TRC Cloning Vector Protocols Lentiviral Vector Production...
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