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We narrowed to 1,541 results for: ins

Showing: 181 - 200 of 1541 results

  1. New CRISPR Tools: Cas7-11 and PASTE

    Type
    Blog Post
    ...bacterial genome, and inserted it into E. coli to express and then test for any proteins that could cut RNA...the lab instead turned to phage serine integrases, where they saw 10-20% integration of inserts up to 36...Schematic of programmable gene insertion with PASTE. The PASTEsystem involves insertion of landing sites via Cas9...single-stranded nicks instead of the double-stranded breaks used in other insertion systems. Off-target ...nonetheless a new subtype. Cas7-11 had combined all the domains into a single protein, shedding the complexity ...Cas7-11   Use The Cas7-11 targeting system contains two pieces: a single protein, computationally ...computationally described as a fusion of four Cas7 proteins and a putative Cas11-like protein, and a guide RNA. The ...

  2. Viral Vectors 101: Parts of the AAV Packaging Plasmid

    Type
    Blog Post
    ...Rep78 and Rep68 contains a DNA-binding and endonuclease domains, all four Rep proteins share a common ...which contains the AAV structural and packaging genes, the adenoviral helper plasmid which contains the ...the proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome...Rep proteins. Alternative splicing generates the different C-terminal regions of the Rep proteins. While...common helicase and ATPase domains. To function, the Rep proteins form oligomers of Rep 78/68 or Rep52/40. ... start sites generate the three VP proteins. The three VP proteins assemble into capsid at a 1:1:10 ratio...gene of interest can be packaged into the virus instead.  Next let’s take a closer look at the rep and ...

  3. Visualizing Protein Turnover In Situ

    Type
    Blog Post
    ...incorporated into newly synthesized proteins instead of methionine. The labeled proteins can be detected using click-chemistry...turnover of individual proteins in situ.  For the visualization of specific proteins in situ immunocytochemistry...audiences. Right now, she is a science instructor at The Innovation Institute in Boston. When Eugenia is not ...protein is decreased in Alzheimer’s disease patient’s brains. However, it is not known why or how this happens...degradation. These processes control the abundance of proteins in cells. Homeostatic regulation of protein abundance...type. However, we cannot distinguish new from old proteins within that pool. Therefore, it is not possible...used to facilitate the visualization of specific proteins in cells. Tagged protein can easily be overexpressed...

  4. Uncertainty about Labor Law Brings More Uncertainty to Postdoc Wages

    Type
    Blog Post
    ...were at institutions focused only on tracking hours, and 22% of postdocs were at institutions where no... of some public institutions) to cancel plans to raise salaries. What are institutions doing after the...planning unionization at their institutions. One clear message that institutions have sent, with the lack of...their institution is doing from Future of Research over social media, not from their institution), is ...raise were: postdocs at institutions like Stanford and the Whitehead Institute, where salaries had already...street from these institutions) are raising salaries. Postdocs at the Salk Institute in La Jolla California...This post discusses how a nationwide injunction against the FLSA is affecting universities' decisions to...

  5. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    ...editing techniques. Inbred and outbred strains Mouse strains used in research can fall under one of two.... Common inbred strains include C57BL/6, Balb/c, and 129. Conversely, outbred strains were originally ...Examples of outbred strains include Swiss Webster and Harlan (Hsd) National Institutes of Health (NIH) Swiss...broad classifications: inbred or outbred. Inbred strains share a common genetic background, which means ...limited but genetically diverse gene pool. Outbred strains are strategically bred to maintain maximum genetic...genetic diversity of the human population. Outbred strains of mice are useful for comparing dominant and recessive...Genetically engineered mice Mice can be engineered by inserting a targeting vector containing the gene of interest...

  6. The Golden Stopwatch Award: Recognizing Facilitators of Research

    Type
    Blog Post
    ...Hospital  Brandeis University  Broad Institute  California Institute of Technology Cold Spring Harbor Laboratory...Laboratory  Dana-Farber Cancer Institute  Fred Hutchinson Cancer Research Center Memorial Sloan-Kettering...  National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK)  National Institute of Genetics...Rockefeller University  Salk Institute for Biological Studies The Scripps Research Institute St. Jude Children's...University and Medical Center  Weizmann Institute of Science Whitehead Institute for Biomedical Research Glossary...intellectual property, and to help requesting institutions safeguard themselves from undue liability. Before...materials? If so, you should always talk to your institution’s TTO or legal office first. Generally, Addgene...

  7. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...interest. Tagged proteins are typically expressed at much higher levels than endogenous proteins. Data obtained...Immunoprecipitation (IP) uses immobilized antibodies, or immunoglobulins, to isolate a specific protein out of a complex...study how a target protein interacts with other proteins or nucleic acids. Read on to learn more about ...optimized strategies for differing amounts of target proteins or antibody bonding strengths.    . Steps...from cells or tissues contain a complex mix of proteins seen as green squares, purple circles and blue...collected by centrifugation or a magnet. 4) Unbound proteins are washed away. 5) The target protein is eluted...eluted.     In the first method, an antibody against a target protein is immobilized, or tethered, on...

  8. Antibodies 101: Single Chain Fragment Variables (scFvs)

    Type
    Blog Post
    ...generations of scFvs against two antigens that are impossible to generate scFvs against in vivo: antigens...the sequences for the variable domains of the selected scFvs are inserted into a vector encoding the antibody... are often the building blocks for engineering proteins. So let’s get acquainted with scFvs. What is an...variable heavy chain (VH) of an antibody. These two chains are connected by a flexible linker peptide that...scFv. While you might assume that the variable domains of an scFv would mirror that of an antibody (VL-linker-VH...antigens found in the body or self antigens, and toxins that are lethal to animals.  In the clinic, scFvs’ size...to produce scFvs in mammalian, yeast, plant, and insect cells, they are most often expressed from a plasmid...

  9. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

    Type
    Blog Post
    ...interactions as well as be used for both soluble and insoluble proteins, which can be a challenge for classic antibody...can take hours to label proteins and APEX2, although it can rapidly label proteins, requires H2O2 which ...interacting proteins in one simple experiment. PCA is an in vivo technique where two proteins are fused ...interaction of two proteins while also biotin labeling and purifying additional proteins from a complex of... and an RNA component that contains an RNA motif of interest. Any proteins that bind the RNA motif will...interacting cell surface proteins with a biotin tagged LPETG peptide. Biotin tagged proteins can then be purified...There are approximately 2-4 million proteins per cubic micron in bacteria, yeast, and mammalian cells...

  10. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    ...ankyrin repeat proteins, or DARPins, are genetically engineered small affinity proteins, around 14-15 ...antibodies. Instead, they are derived from a class of binding proteins known as ankyrin repeat proteins. DARPins...and heavy chains made by immune systems. But here, we're not interested in antibodies. Instead, we're going...engineered fusion proteins similar to a Fab fragment but created through engineering instead of cleavage. ...synthetic binding proteins which use the Fibronectin type III domain as their scaffolding instead of the antibody...the ability to specifically recognize and bind proteins. They’re used in a number of research and clinical...these alternative reagents are created in the lab, instead of generated in vivo, they have higher reproducibility...

  11. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ... the presence of proteins from samples containing a mixture of proteins. The proteins are separated based... Antibodies, also known as immunoglobulins, are ~150 kDa, Y-shaped proteins that are both a natural part...region. Two immunoglobulin (Ig) heavy chains and two Ig light chains make up each antibody molecule. The...the Ig heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable...Flow cytometry is used to detect proteins on the surface of or inside whole cells. Antibodies - typically...antibodies are produced by B cells. They bind to proteins on the surface of extracellular pathogens such...such as parasites or microbes, or to proteins expressed on the surface of cells that have been infected ...

  12. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...Margaret Robinson’s lab, and more recently used by Stephen Royle’s lab, acutely depleted proteins from their...induction heterodimerization of proteins that contain rapamycin-binding domains, such as FKBP and FRB. The ...inactivation. Find the Robinson plasmids or the Royle plasmids and get to work! Robinson et al., Dev Cell...light-oxygen-voltage (LOV) sensing domains from algae into chimeric Opto-RTKs. The LOV domains bind flavin cofactors...phosphoserine (SP218) insertion was moderately enhanced while two phosphoserine insertions (SP218/SP222) was... to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other markers...demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the genome. Although...

  13. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    ...working with purified proteins in vitro, you can even use the same tag for multiple proteins but label each ..., V. W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research...protein of interest tagged with different fusion proteins for different experiments over the years — a green...been mutated to block its catalytic activity and instead become trapped in a covalent bond with its substrate... dyes provide many advantages over fluorescent proteins, opening up new applications.   Figure...Griffin et al., 1998), this 15 amino acid peptide contains a cluster of four cysteines (CCXXCC, where X is... Michael Sheetz labs demonstrated in 2005 that proteins fused with E. coli dihydrofolate reductase (eDHFR...

  14. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...mix of proteins with a broad size range to be separated on a single gel.  Immobilizing proteins on a membrane...albumin or other proteins. The sticky areas on the membrane will bind to the proteins in the buffer, blocking...labeled. Instead, a conjugated species-specific secondary antibody is used to visualize the proteins. In this...Introduction to western blotting  In a western, proteins are: (1) separated by size, (2) transferred to...absence of a protein, but can also determine if proteins are being up- or downregulated in a system, detect... to standards, detect the cellular location of proteins, and can be a readout for protein interaction ...applied to the gel causes the negatively charged proteins to migrate toward the positive charge at the bottom...

  15. INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

    Type
    Blog Post
    ...manner to Type I-F, but instead employ a Cas12k protein instead of three Cas proteins (Fig. 1A, bottom), and...transposase proteins (TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon), which contains the DNA cargo...programmability: depending on the transposases involved, they insert either randomly, or at fixed genomic target sites...generation of tools for efficient and programmable insertions of large DNA cargos into bacterial genomes. This... This new capability was named INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting...spacer sequence specifying the target site. Four proteins (TniQ, Cas8, Cas7, Cas6) that forms the QCascade...INTEGRATE. The requirement for multiple Cas proteins to form the Cascade targeting complex is a hallmark...

  16. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    ... prey proteins after screening. It is also possible to screen libraries of bait and prey against each ... expression of target proteins in heterologous systems (e.g. mammalian proteins in S. cerevisiae). ...activation and cell biology. During this period, proteins were successfully expressed as fusions that retain...domain (DBD) and transcriptional activation (TA) domains retain their individual activities when separated...could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown is still ...also called the interaction trap), genes encoding proteins of interest(POI’s) are fused to TAs and DBDs (... then co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter...

  17. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

    Type
    Blog Post
    ...contain your insert of interest. Simply stated, ccdB makes cloning easier by selecting against vectors that...vector “backbone” in this system contains ccdB. A successful insertion will completely replace ccdB with...vectors in mind, any plasmid that contains the F plasmid (F’ strains) will also be resistant to CcdB, ...popular lab strains, such as NEB Stable, JM109, XL1 Blue or XL10 Gold, that are F’. These strains could possibly...that did not take up your insert. But exactly how does ccdB accomplish this? Let’s start with a brief ...antitoxin protein (CcdA) that protects the cell against the toxic CcdB. Cells that lose ccdA through the..., an investigator were to successfully clone an insert into the vector, the ccdB reading frame would be...

  18. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ... yeast overexpression vector. This plasmid contains origins of replication and selectable markers that...the S. cerevisiae met deletion strains and then isolate transformed strains on media that lack uracil, but...function wide open. What if two seemingly similar proteins have acquired divergent functions due to mutations...research questions. The idea is straightforward. Instructors first select a multi-gene pathway to study and...and a reference organism where deletion strains are available. Then, staff or students clone similar sequences...overexpression plasmids. Students next transform deletion strains from the reference organisms with the overexpression...addition, a complete set of well-annotated deletion strains is available (6). Methionine synthesis in S. cerevisiae...

  19. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...for each insertion. Also, it remains challenging to screen for the genetically modified strains that contain...tagging proteins in a tissue specific manner, as well as 3-site destination vectors for inserting a tag ...tagging endogenous proteins with an auxin-inducible degron (AID) tag to generate proteins which can be rapidly...years ago, the growing spectrum of fluorescent proteins (FPs) has been an invaluable resource for studying...with FPs. Overexpression of fluorescently tagged proteins can lead to improper protein localization, protein...new user-defined DNA sequence (like GFP) can be inserted at the DSB. CRISPR has allowed scientists to tag...contain your newly tagged gene. Most insertions can only be detected by labor-intensive processes like...

  20. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...sorting or FACS (2). By analyzing single cells instead of the population as a whole, scientists gain statistical...Techniques is Best for Me?  An overview of the instrumentation A flow cytometer combines three systems to ...receptor are stained with a fluorescent antibody against the receptor and analyzed on a flow cytometer. ...cells pass through the path of a laser and the instrument collects information about the cell’s size, complexity...targets. If you are planning on using fluorescent proteins for your experiment and are unsure which to choose...Fluorescent Protein Should I Use? However, fusion proteins tag your protein of interest with a fluorescent...C-terminus of the protein. One downside of fusion proteins is that the tag can alter the structure of the...
Showing: 181 - 200 of 1541 results