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We narrowed to 833 results for: ins

Showing: 101 - 120 of 833 results

  1. Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

    Type
    Blog Post
    Published
    June 15, 2017, 2:30 p.m.
    ..., the red fluorescent proteins form tetramers. In general, fluorescent proteins have a natural affinity...true monomeric fluorescent proteins. Concluding remarks Many fluorescent proteins are available, and their...aggregation The tendency of purified fluorescent proteins to form homodimers or higher order oligomers can...-PAGE under non-denaturing conditions separate proteins based on size. Whether higher molecular weight...protein, reflect the likelihood that fluorescent proteins interact or oligomerize in cells? This question...question is largely unanswered. Ideally, fluorescent proteins should act as inert modules that fail to homodimerize...homodimerize in cells. To understand how well fluorescent proteins approach this ideal situation, cellular assays...

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

    Type
    Blog Post
    Published
    Dec. 3, 2020, 2:15 p.m.
    ...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. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    Published
    July 11, 2019, 1:26 p.m.
    ...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...

  4. The Golden Stopwatch Award: Recognizing Facilitators of Research

    Type
    Blog Post
    Published
    Sept. 5, 2019, 1:06 p.m.
    ...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...

  5. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ...could be affected by the location of insertion. Lentiviral insertion can also disrupt the expression of ...Low Efficient and Stable Genome integration Insertional mutagenesis, transgene reactivation, residual...Low Efficient and Stable Genome integration Insertional mutagenesis, residual expression piggyBac ... inefficient Difficult to purify reprogramming proteins. Table 1: Key Features of Different Methods...of iPSC generating methods due to the risk of insertional mutagenesis. Random integration also creates ...potential. Finally, retroviruses are prone to insertional mutagenesis and random integration creates variation...Addgene. Another drawback is that lentiviral vectors insert in different genetic locations in each transduced...

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

    Type
    Blog Post
    Published
    June 3, 2021, 1:15 p.m.
    ...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...

  7. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    Published
    April 14, 2022, 1:15 p.m.
    ...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...

  8. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    Published
    Dec. 15, 2016, 3:57 p.m.
    ...  The best applications for dsDNA inserts include: large insertions or deletions, including selectable...note* not all linked strains contain the Lambda Red system).  In some of these strains, expression of exo... used to make an assortment of modifications: insertion and deletion of selectable and non-selectable ...of the experiment. dsDNA substrate is best for insertions or deletions greater than approximately 20 nucleotides... only a few base pairs.  dsDNA Substrate dsDNA inserts can be made by PCR using primers that amplify the... with 50 base pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically...not determine the direction of replication and instead design oligos that target both strands. One of ...

  9. Plasmids 101: Optimizing Plasmid Yields

    Type
    Blog Post
    Published
    May 26, 2016, 2:30 p.m.
    ...high quality DNA that contains these elements. Be aware of endA+ strains Strains like Stbl3 have been ...incomplete. Protein expression strains are not good for cloning Bacterial strains adapted for protein purification...bacterial strain Differences between E. coli host strains can impact plasmid yield. Check the bacterial strain...an example). Look out for carbohydrates Certain strains, including HB101 and derivatives, release large...have previously reviewed many common lab E. coli strains on the blog, and also recommend this handy guide...for a more extensive listing of common E. coli strains. Increase the volume Starting with a larger culture... be affected by the plasmid size, nature of the insert, propagation strain, growth conditions, and the...

  10. Plasmids 101: Protein tags

    Type
    Blog Post
    Published
    Dec. 11, 2014, 4:26 p.m.
    ...tags and fusion proteins to aid in the expression and purification of recombinant proteins. Tags for stability...recombinant proteins for one-step purification with glutathione, can also protect against proteolysis....one form of instability. Prokaryotes can also have a hard time folding eukaryotic proteins. You can get...101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection... biologists who need to overexpress and purify proteins can face any number of technical challenges depending...protein (MBP). Overexpression can also lead to insolubility, and aggregated protein is not useful protein... but scientists may also choose to add smaller proteins, such as Thioredoxin A (TrxA) that improve disulfide...

  11. A Practical Approach to Choosing the B(right)est Fluorescent Protein

    Type
    Blog Post
    Published
    June 1, 2017, 2:30 p.m.
    ... of posts explains how to critically assess the reported properties of fluorescent proteins, how to do...task. Many fluorescent proteins are available and the number of fluorescent proteins is steadily increasing...Before you start a new project with fluorescent proteins, the best advice is to try a couple of promising...do a head-to-head comparison of fluorescent proteins and how to make a well-informed decision on the best...data that summarizes properties of fluorescent proteins on websites. To make the best choice, the fluorescent...relevant conditions. The properties of fluorescent proteins are usually only examined under a few conditions...and provides a quick way to compare fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al...

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

    Type
    Blog Post
    Published
    April 16, 2024, 1:15 p.m.
    ...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...

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

    Type
    Blog Post
    Published
    May 22, 2018, 1:38 p.m.
    ... 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...

  14. Alex Chavez on MD PhDs, CRISPR, Gene Drives, & More

    Type
    Blog Post
    Published
    Nov. 15, 2016, 3:30 p.m.
    ...Fellow in the Church and Collins labs at the Wyss institute for Biologically Inspired Engineering. Alex is...Listen to this episode for insights into the process of getting an MD PhD, the inside scoop on developing new...postdoc 12:01 - 13:21 : The Wyss Institute for Biologically Inspired Engineering and why Alex decided ...mutant microbial strains faster than ever before. 29:54 - 31:25 : Nature's defenses against gene drives. ...Page. 2:00 - 3:55 : Alex introduces himself and explains why he decided to do an MD PhD - it's all in the...- 7:04 : Alex describes the MD PhD process and explains why he loved the first 2 years of medical school.... 13:22 - 22:07 : Alex's projects at the Wyss Institute - New CRISPR tools and the importance of moving...

  15. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    Published
    Sept. 14, 2016, 2:30 p.m.
    ...binding proteins in as little as 3 days. Abil et al., 2014 show that you can fuse custom PUF proteins to a...you’re an academic researcher at a nonprofit institution, you can order plasmids covering a wide range...expression in bacteria, yeast, mammalian cells, insects, and even Xenopus oocytes. The expression vectors...antibiotic efflux pumps and ankyrin seven-repeat proteins: Sjuts, Hanno, et al. "Molecular basis for inhibition... from affecting its translation initiation and instead makes initiation heavily dependent upon the core...Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium... "Modular Synthetic Inverters from Zinc Finger Proteins and Small RNAs." PloS one 11.2 (2016): e0149483...

  16. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    Published
    April 19, 2016, 2:30 p.m.
    ...with dimerizing fluorescent proteins is illustrated. 1) Membrane proteins fused to FPs that dimerize can... National Institutes of Health and he is currently an Associate Professor at Albert Einstein College of...-based FRET, with application to intrinsically unstructured proteins. Protein Sci, 2007. 16(7): p. 1429... Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal...(i.e. DsRed) [3]. This is a problem for fusion proteins. One of the major applications of FPs is to visualize...Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors and transporters, are integrated... to an obligate dimer or oligomer Several cell proteins normally self-associate into homodimers or even...

  17. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    Published
    July 20, 2021, 1:15 p.m.
    ...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...

  18. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    Published
    Sept. 8, 2016, 2:30 p.m.
    ... Georgeta, and Justin W. Taraska. "Imaging proteins inside cells with fluorescent tags." Trends in biotechnology...Janelia Research Campus, Howard Hughes Medical Institute. Chemistry is dead, long live chemistry! The discovery... their own. Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive...budgets are a widespread issue with fluorescent proteins—even the thriftiest cell biologist can feel like...brighter and more photostable than fluorescent proteins, providing a straightforward way to improve the...combined with the ease and specificity of fluorescent proteins. In-cell labeling strategies The majority of ...tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure 1a). Other strategies that have...

  19. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    Published
    Jan. 10, 2023, 2:15 p.m.
    ...occurring E. coli plasmid ColE1 (Goebel & Helinski, 1968; Bazaral & Helinski, 1968), in which the plasmid was ...1981). E. coli strains such as DH5α, HB101, NEB Stable, Stbl3, etc., are recA- mutant strains, and therefore... of plasmids, taking many forms (Levene, 2009; Higgins & Vologodskii, 2016). Plasmid multimerization occurs...to occur more frequently in plasmids with large inserts (>7 kb)(Berg et al., 1989), direct repeats (Ribeiro...et al., 2009). Since multimers have additional origins of replication compared to monomers, they are also... of multimer present in your sample. In these instances, please note that the supercoiled dimer band can.... If the presence of a multimer in your sample remains unclear after visualizing it on a gel, one of the...

  20. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    Published
    Feb. 10, 2016, 3:30 p.m.
    ...Mutations Insertions, deletions, point mutations Insertions, deletions, point mutations Insertions, deletions...-4 phage proteins are needed and these can be provided via a separate plasmid or be inserted into the ...recombineering proteins Red/ET from a temperature-sensitive plasmid. Homologous recombination then joins the linear...successfully incorporated a 60-nucleotide insertion; beyond this insertion size, primer synthesis is more expensive...making small point mutations, large deletions/insertions require complicated, often costly in vitro assembly...the power of bacterial recombineering to create insertions, deletions, and substitutions - at the same efficiency...colonies carrying the unmodified plasmid. For insertions, it’s possible to use the same technique just...
Showing: 101 - 120 of 833 results