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We narrowed to 584 results for: abo.1

Showing: 81 - 100 of 584 results

  1. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    Published
    March 15, 2016, 2:30 p.m.
    ...type of assay is a clonal cell line known to contain 1 integrated copy of the qPCR target gene. Developing... successful transduction experiment. References 1. Kutner, Robert H., Xian-Yang Zhang, and Jakob Reiser...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009... titration methods."BMC biotechnology 6.1 (2006): 1. PubMed PMID: 16836756. PubMed Central PMCID: PMC1534021...events." Genetic vaccines and therapy 2.1 (2004): 1. PubMed PMID: 15291957. PubMed Central PMCID: PMC514534...drawback of this approach is that it can be quite labor intensive. Target cells are transduced with serial...

  2. Technical Design of a Western Blot

    Type
    Blog Post
    Published
    Aug. 22, 2024, 1:15 p.m.
    ...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...

  3. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    Published
    Aug. 4, 2016, 2:30 p.m.
    ...the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...it was first published in Nat Biotech in May 2016 (1), NgAgo has received much attention and the plasmid...Pooran's NgAgo Survey Survey results As of August 1, 2016, a total of 165 researchers responded to the...the cell, the same method used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's... dreams of a sunny day in Edinburgh.   References 1. Gao, Feng, et al. "DNA-guided genome editing using...technology. To dig more into what researchers felt about NgAgo, I conducted a survey and asked researchers...successful, and the second section asked basic questions about the NgAgo protocols used by respondents (transfection...

  4. Finding and Joining Your Dream Lab

    Type
    Blog Post
    Published
    April 8, 2014, 2:50 p.m.
    ...choosing direction of a project. If you expect more 1:1 time with the advisor, you should choose accordingly...hire visits the lab for a full day, gives a 1 hour talk about his or her interests, and spends one-on-one...that supports both paths – you won't know in year 1 what you will want in year 6. Have lab alumni taken...during the day? We asked three lab heads how they go about selecting new hires. This is followed by some perhaps...current members to share short-term goals and chat about awesome science. Important factors in the search..., the lab has a reputation for not entering collaborations. Alternatively, perhaps the lab publishes and...Medical School) for taking the time to speak with us about adding new scientists to their labs. Read the Other...

  5. Antibodies 101: Monoclonal Antibodies

    Type
    Blog Post
    Published
    June 29, 2021, 1:15 p.m.
    ...animal, those B cells are fused to myeloma cells at a 1:1 ratio and then cultured in a special medium called...antibodies that recognize proteins. Figure 1: Steps in the generation of hybridomas for monoclonal...Washington (DC): National Academies Press (US); 1999. 1, Generation of Hybridomas: Permanent Cell Lines Secreting... Learn more about research applications for antibodies in Intro to Antibodies Read about the Developmental...antibodies in your experiments, you may be curious about all of the different kinds of antibodies that are...Disadvantages of monoclonal antibodies As mentioned above, genetic drift is one concern when using hybridomas...Hybridoma Bank, a repository for hybridomas  Learn about monoclonal antibodies for immunotherapy Resource...

  6. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    Published
    June 23, 2016, 2:30 p.m.
    ...cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation...three components required for translation (Figure 1): A tRNA that recognizes a specific ‘open’ codon ... open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...modification allowed the deletion of release factor 1, the protein responsible for terminating translation...Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing...follow her on Twitter @nieminm.   References 1. Cohen, Philip. "The origins of protein phosphorylation." Nature... one of the MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic...

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

    Type
    Blog Post
    Published
    July 26, 2018, 12:46 p.m.
    ...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...which have previously been characterized. See Box 1 in Kudo et al. for more details on the design of the...using translocation reporters. Nature protocols, 13 1, 155-169. PMID: 29266096. Find the KTR plasmids here...Blog Learn about photoswitchable kinases Learn more about fluorescent biosensors Learn about using phosphoserine...

  8. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ...two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or...with linearized vector backbone. DpnI digestion - 1 μl DpnI is added to the PCR mix to remove parental...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ... Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential...with successful recombination reported in all laboratory E. coli strains tested so far. The use of this...

  9. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    Published
    Feb. 27, 2018, 2:04 p.m.
    ...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit and -...dyes are also commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is...Control Protein Activity Learn more about protein tags Read about E. coli strains for protein expression..., if not better, than antibodies for all of the above listed molecular techniques, but they can also be...protein purification methods. Read on to learn more about nanobodies and how their structure and function ...

  10. Common Injection Routes in Mice

    Type
    Blog Post
    Published
    Jan. 22, 2025, 9:42 p.m.
    ... advanced and finally, to expert.   Figure 1. Visual representation of common injection routes ...back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill required: beginner Intradermal ...work. Good luck with your in vivo experiments! Table 1: Summary of common injection routes in mice. Injection...hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia Between...resources on the Addgene blog Mouse Modeling Part 1 Mouse Modeling Part 2 Five Popular Model Organisms...Routes of administration. In H. Hedrich (Ed.), The Laboratory Mouse. Academic Press. Turner, P. V., Brabb, ...M. A. (2011). Administration of substances to laboratory animals: routes of administration and factors...

  11. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    Published
    Jan. 10, 2017, 3:37 p.m.
    ...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...of the Addgene Scientific Advisory Board. His laboratory at Columbia University is focused advancing scientific... Listen to Our Interview with Alex Chavez Learn about Sequencing Options for CRISPR Genotyping Cas9 Activators...

  12. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    Published
    Aug. 30, 2022, 1:15 p.m.
    ...preventing your primary antibody from binding (Fig.1). The level of cross-linking that occurs depends on...proteins, which could disrupt your target epitope (Fig. 1). So, while organic solvents may improve antibody ...thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26  Rolls G (2022) Process of Tissue Fixation...www.leicabiosystems.com/us/knowledge-pathway/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives...and detergents.  Organic Solvents  As mentioned above, organic solvents can be used to simultaneously ...permeabilizing with saponin. However, if you are not worried about washing away your target (for example, some cytoskeletal...

  13. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    Published
    April 20, 2021, 2:30 p.m.
    ...disadvantages: Table 1 (Joglekar et al., 2017)  Table 1 (Gutierrez-Guerrero et al., 2020) Table 1 (Cronin et al...polyA tail.   Figure 1: Lentiviral production uses three plasmids: (1) The transfer plasmid, (2)... specific purposes as described previously (Table 1, Gutierrez-Guerrero, et al, 2020). Because retrovirus... also toxic at high concentrations as described above and is inactivated by the complement system in serum...

  14. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ...the sample (by less than 1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and...since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli...that could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV while...our tissues unimpeded. Andrew and Maria dreamed about the possibilities of a magnetoresponsive fluorescent...of improved response. Then, just when Andrew was about to suggest she deprioritize the project, Maria’s...with a strong commitment to exploration and collaboration. Andrew is a physicist with a background in ...

  15. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    Published
    April 6, 2023, 3 p.m.
    ..., rather than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse IgG2a secondary would...IgG2a>IgG2b>IgG1 (Manning 2012).      Figure 1: Broadly reactive versus subclass-specific antibodies...antibody binding and therefore the signal strength (Fig. 1). Monoclonal primaries If you do decide on a monoclonal...raised in a different species.      Table 1: Isotype considerations when choosing antibodies ...Elsevier. https://doi.org/10.1016/s0091-679x(08)60255-1  Manning, C. F., Bundros, A. M., & Trimmer, J. S. ...In a direct approach, that is simple - but what about when you need to use an indirect (primary and secondary...

  16. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    Published
    Dec. 22, 2022, 2:15 p.m.
    ... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...before attempting knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break...traditionally have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently...breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S....CRISPR Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to ... post on increasing HDR efficiency to read more about optimizing this distance. Homology arms and types...screening strategies for your experiment. Also learn about Addgene’s knock-in plasmids for reagents to help...

  17. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Oct. 14, 2015, 2:30 p.m.
    ... more about Cpf1 and check out our CRISPR guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1... than the multi-subunit complex employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided...mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but with the direct repeat... Cpf1 was first identified, we didn't know much about its on-target and off-target editing efficiency....

  18. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    Published
    March 14, 2017, 2:30 p.m.
    ...such as IL-8, IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit regulatory...introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector...the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF to increase...This study used CRISPR gene editing to knockout PD-1 on immune cells harvested from patients and thereby...exploring new avenues in healthcare.   References 1. Vinay, Dass S., et al. "Immune evasion in cancer:...lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial." The Lancet 385.9967 (2015)...potential beneficiaries. Collaborative therapy For the most efficient outcomes, collaborative therapies must be...

  19. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    Published
    July 2, 2020, 12:40 p.m.
    ...questions about chromatin conformations. The Xu lab presented three ways to use CAPTURE 2.0: 1. Multiplex...by PCR or next-generation sequencing. Figure 1: Overview of the key steps of the CAPTURE method. ...ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or ...or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs...(s) Cell lines Cell lines, primary cells Table 1: Key differences between original CAPTURE and CAPTURE...with current 3C methods provide useful information about chromatin interactions, they are grainy so it’s ...Cas9 Variant for Your Next Experiment Learn More About Designing gRNAs Against Your Favorite CREs Here ...

  20. Early Career Researcher Toolbox: Social Media for Scientists

    Type
    Blog Post
    Published
    Dec. 11, 2018, 12:46 p.m.
    ...blog post about a research paper that explored how scientists connect on Twitter that I realized 1) a lot...engage with others about your topic(s) of interest. Hopefully through these links, I’ve: 1) convinced you ... but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn... have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast...saw a request for an Addgene guest blogger post about colony PCR, I signed up immediately. I had used ...science communication skills, while also learning about new scientific developments and helping scientists... found this paper. I wrote an Addgene blog post about it and shared the paper with my lab since one of...
Showing: 81 - 100 of 584 results