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We narrowed to 1,025 results for: URE

Showing: 151 - 180 of 1025 results

  1. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ...packaging as a single payload (Figure 4). Figure 4: ONE-GO biosensors measure the formation of Gα-GTP...uses new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced...amplification of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-...heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https://doi.org...N-terminal CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression...solution and when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule... inserted. B) Protein expression and labeling procedure. Adapted from Smith et al. 2024 under a CC-BY ...

  2. A Guide to Starting Your Own Journal Club

    Type
    Blog Post
    ...walks through the paper figure by figure. Some follow the same figure by figure organization with a different...adapt the second version (multiple presenter, figure by figure) if the primary purpose is training graduate...enjoy is discussion of current new and exciting literature. As I transitioned out of academia and away from...researchers of all varieties must stay current with literature and emerging technologies to remain competitive...in journal clubs. They also have likely had the pleasure of attending both productive and not so productive...pieces before that first meeting takes place to ensure success. One of the most important is letting people...ready for your first journal club to begin! Figure 1: Discussing next-generation sequencing at the...

  3. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    ...strategies such as the bisarsenical dye (Figure 1a) and FAP systems (Figure 1e) are inherently fluorogenic and...biological environments such as live cells and tissues (Figure 1). These flexible strategies give you the best...peptide tag could be used to label proteins in cells (Figure 1a). Other strategies that have been developed ...substrate ligand motif attached to a fluorophore (Figure 1b). Engineered ligases (e.g., lipoic acid ligase...attachment of a fluorophore ligand to a peptide tag (Figure 1c). Click chemistry (e.g., transcyclooctene–...amino acids can be incorporated into a protein structure and then used with the growing toolbox of bioorthogonal...fluorophore at the specific site of incorporation (Figure 1d). Fluorogen activating proteins (FAPs) – These...

  4. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ... need to pick a fluorophore for each target and ensure the fluorophores, together, are able to give you...most efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining...Fluorescence Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective colors of a ... we can recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE and ...laser. To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488...maximum of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at...relatively stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The ...

  5. Hot Plasmids Spring 2024

    Type
    Blog Post
    ...transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the ...recommends both for use in western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-...improvements in performance.      Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor...reconstituted within the target cells (Figure 6).   Figure 6: Illustration of split fluorophore ...Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal ... that incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in...just begun with PE7, it will be exciting to see future studies enabled by this tool as well as additional...

  6. What's New in CRISPR - November 2021

    Type
    Blog Post
    ...will cover: Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas...problem. A miniature CRISPR-Cas system for mammalian use Stanley Qi’s lab has developed a miniature Cas system...paper in Nature Biotechnology Find the Cas12f-GE plasmids Programmed genome editing by a miniature CRISPR-Cas12f... guide RNAs Yong-Sam Kim’s lab also created a miniature yet active Cas system starting with the same Un1Cas12f1...nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes a Cas12f from a different...ribonucleoprotein, and AAV.  Read the paper in Nature Chemical Biology Find the AsCas12f1 plasmids   ...paper in Science Find the SEND plasmids    Figure 1: SEND combines an endogenous Gag homolog, cargo...

  7. Fluorescent Proteins 101: Fluorescent Protein Timers

    Type
    Blog Post
    ...and, when they mature, fluoresce red. The ratio of the mature color (red) to the immature color (green ...determination of expression time The ratio of mature to immature fluorescence from the FP timer is dependent...their knowledge of protein structure and saturation mutagenesis. mK-GO matures from green to red, and the...they predominantly fluoresce one color but slowly mature until they predominantly fluoresce a second color...580 nm) over the course of 18 hours in vitro (see figure 1B in Terskikh et al.) and even displays predictable...linear window for the theoretical timer shown in Figure 1B, you can use the equation fit to this linear... system composed of two separate FPs (one that matures quickly and the other slowly, see Verkhusha et ...

  8. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ... from a heterogeneous mixture - for example, cell lysate or media from cultured cells. ELISA can be used...cell subsets of interest from among the mixture and to measure the relative amount of your protein of ...antigen the antibody recognizes and binds to.   Figure 1: Labeled diagram of an antibody including Fc,...). Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv...Chain Fragment Variables (scFvs). Nanobodies Figure 3: Comparison of the Hcab and nanobody. Nanobodies...can be used for qualitative and quantitative measurement of protein expression in cell lysates, whole ...presence of proteins from samples containing a mixture of proteins. The proteins are separated based on...

  9. Antibodies 101: Multiplex Immunofluorescence

    Type
    Blog Post
    ... IF instead (Figure 1B). To label all your targets in one step, you simply use a mixture of primary antibodies...puncta in each cell. When making figures for a paper or presentation, make sure the images are accessible to...as immunocytochemistry (ICC) when the sample is cultured cells or immunohistochemistry (IHC) with tissues... you can perform simultaneous detection with a mixture of primary antibodies and appropriate secondary...(Im et al., 2019; McLaughlin, 2019).    Figure 1: Schematic of multiplex immunofluorescence approaches.... Sequential IF For a sequential IF experiment (Figure 1A), you’ll block, add primary antibody to bind...antibodies, wash, add a mixture of secondary antibodies, and image all the different colors in one session...

  10. Plasmids 101: Control Plasmids

    Type
    Blog Post
    ...is consistent with the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal...effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid B (as positive control...control plasmids? Generally, control plasmids help to ensure that the observed phenomenon in your experiment...of Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown...vector and internal control A transfection control measures transfection efficiency and enables observation...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore...transfection control is an internal control vector, which measures transfection efficiency. An internal control may...

  11. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...the full plasmid and confirm features you annotate on this page. Figure 7: Enter information about the... (for emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit...are two options to begin your plasmid deposit. Figure 2: Choose to submit plasmid(s) online or by using...deposits of 10 or more plasmids.       Figure 3: If submitting plasmid(s) online (left), choose...will become your article title.       Figure 4: Add plasmids to the article by clicking “Add...begin entering the specifics of your plasmids. Figure 5: Click the "Enter Data" button to add your plasmid...Continue to Next Step" (blue button at the bottom of Figure 6) or you can click the "Save" button and jump ...

  12. Plasmids 101: Walkthrough of Addgene’s Snapgene-Powered Quality Control Process

    Type
    Blog Post
    ... contain similar features and it allows you to more quickly identify the same feature in other plasmids...our new Snapgene generated maps provide improved feature detection with an easy to use interface. We regularly...quality control process because of its expansive feature library and useful tools. In this blog, we’ll walk... a plasmid and we’ll highlight some of the new features available on our website through our Snapgene ...quality control are: To identify important plasmid features To align NGS sequencing results with reference...further plasmid verification Identifying plasmid features While our quality control process for incoming...Snapgene to visualize the sequence and look for features. The first thing that the Snapgene software will...

  13. Fluorescent Proteins 101: Introduction to FRET

    Type
    Blog Post
    ...nanometers of each other, or measure the tension in the helical structure of spider silk, or the activity...that you want to measure (overexpressing a gene or protein after transfection). Future of FRET The first...in a synapse. What kinds of tools enable us to measure these properties, and what fascinating experiments...dipole-dipole interaction. The efficiency of FRET can be measured for a given donor-acceptor pair and a change in...fluorophores and serves as a small-scale ruler to measure distances that are too miniscule for conventional...improved dynamic range for fluorescence lifetime measurements." PloS one 10.8 (2015): e0134436. PubMed PMID...proteins can contribute additional noise to the measurement. If the distance or orientation of the donor ...

  14. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    ...processing (Figure 1). The shRNA sequence is transcribed to produce hairpin shRNA structures that are processed...Dicer chops off the loop of hairpin structures to create a mature siRNA duplex. Fun fact! Endogenous miRNA...upstream and downstream genomic sequences (Figure 2B).   Figure 2: A comparison of an shRNA plasmid sequence...mRNA or recruits other silencing proteins. Figure 1: Comparison of shRNA and miRNA processing and...Choosing the plasmid backbone Choosing the promoter Mature siRNAs typically range from 19–22 nucleotides and...target sequence. However, you can’t just use the mature sequence in a plasmid and call it a day. You need...complementary RNA duplex and the hairpin loop sequence (Figure 2A). Since you don’t need additional flanking sequences...

  15. Pooled Library Amplifications

    Type
    Blog Post
    ... and how will you ensure this?). You should also consider if there are any features of the library backbone... estimate transformation efficiency (Figure 2).    Figure 2: Example of a serial dilution to estimate...might be more difficult to transform.    Figure 1: Factors to consider when creating a pooled library...estimate transformation efficiency. Figure used with permission from the Mark Dawson lab Re-transformation...generally electroporated into electrocompetent cells to ensure the highest possible transformation efficiency....Using Stbl4 or other ultra-high efficiency cells ensures that plasmid diversity in the library is maintained.... The specifics of this can be based on the manufacturer’s protocol for transformation and expected transformation...

  16. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    ...autofluorescence extraction feature. Although autofluorescence extraction is an optional feature of spectral flow...that have distinct spectral signatures and a low similarity index. In figure 3, we can see that PerCP and... detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over into the primary...that spectra to use for compensation.     Figure 1: The emission spectra and filters for FITC and...flow since the machine uses the full spectral signature, not just a narrow band, to discern one marker...fluorophore from another within a fully stained sample. Figure 2 is an example of the emission spectra for two...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711...

  17. Twenty Years of Addgene Sharing: CRISPR

    Type
    Blog Post
    ... In Figure 1, you can see cumulative numbers of CRISPR plasmid distribution and deposits (Figures 1A and...although we used different categories (Figure 2C). Note that in Figure 2, the numbers on the X axis refer ...new tools, illustrated in Figure 2, and the longevity of tools seen in Figure 3, indicates that the usefulness...the top ten countries for each group (Figure 4).    Figure 4: Addgene deposits and distribution ...number of labs who have deposited CRISPR plasmids (Figure 1C), and the cumulative number of CRISPR libraries... 100 new CRISPR depositors each year.    Figure 1: Addgene CRISPR distribution and deposits. (A...300,000 CRISPR plasmids and 12,000 CRISPR libraries (Figure 1D). That’s a lot of editing!  CRISPR functions...

  18. R Bodies: Membrane-Rupturing Microscopic Tools

    Type
    Blog Post
    ... through a structured material over a long distance? And can we understand such a structure well enough...that contain an R body-like structure. As the bacteria develop, the structure extends to rip the bacteria...’t be overstated, we can also learn a lot from Nature’s simpler solutions to transmitting forces over ...contract in response to changing pH, and they can rupture E. coli spheroplasts to release encapsulated proteins...inside cells, and these constructs may be useful to future scientists who would like to further study R body...

  19. Is this the right place for me? 8 tactics for choosing a lab

    Type
    Blog Post
    ...undue pressure, impossible deadlines, unnecessary disruptions Destabilization – including failure to acknowledge...choose and it will make a huge difference for your future career and life. You might see a lab head as choosing...can give open, revealing information about the culture and lab head. While it’s good to talk to new lab...head. Former lab members can give you a fuller picture. Consider talking to people in the lab across the...frequently cancelled? Choosing a role model Make sure the mentors you choose are teaching the stuff you...stay around? Is it too long or do they leave prematurely? Do lab alumni stay in the same field taking...bullying. Even with the rise of movements such as the Future of Research Symposium and #MeTooSTEM, academic ...

  20. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ... users coat a multiwell plate with a capture reagent. The capture reagent may be an antigen or an antibody...et al. originally developed the ELISA assay to measure antibody levels, scientists have since adapted ...on how the assay is set up. Then they block the capture reagent to prevent non-specific interactions. Next...consider alternative detection methods.      Figure 1: The four types of ELISA are direct, indirect...ELISA the multiwell plate is first coated with a capture antibody that binds to the antigen in a sample ...proportional to the level of antigen in the sample. This figure was created with BioRender.com  Direct ELISAs...cross-react with samples, eliminating them from the procedure removes one potential source of cross-reactivity...

  21. CasPEDIA: A Functional Classification of Cas Enzymes

    Type
    Blog Post
    ...give a quick overview of each CasPEDIA feature. Activity features: CasID CasPEDIA will display both the...mixed). This information represents the ‘big picture’ features of the enzymes documented that should narrow...design, etc.). With this feature, you can compare different Cas enzymes to make sure you are getting the desired...resource for the scientific community. What are the features? CasPEDIA has collected relevant information on...Experimental guidelines and applications Protein structure and properties Links to relevant resources The...designed, if that’s your end goal. CasPEDIA also features useful links to Addgene resources throughout the...packaging limitations. Maybe you want to read literature on the less common applications of an enzyme ...

  22. dTAG - You're it!

    Type
    Blog Post
    ...et al., Nature Chemical Biology additionally provides detailed information on cloning procedures used to...at doses as high as 20 µM (see Figures 3 and 5 in Nabet et al., Nature Chemical Biology for examples).... cereblon, leading to rapid target degradation (Figure 1). Plasmids: We generated a series of plasmids...molecules will be commercially available in the near future, and we will provide an update with vendor and ...FKBP12F36V tag without disrupting protein function. A literature search can help determine which terminus is favorable...molecules in your biological assay of choice to ensure that there are no toxicities at the doses used ... roles in Multiple Myeloma), it is important to ensure lack of degradation in your context. Immunoblotting...

  23. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ... photostability measurement of various cyan fluorescent proteins is depicted in figure 1. Of note, the... noted that the photostability measurements reported in the literature are performed in different ways...photostability, an experiment is performed that measures fluorescence intensity over time. In order to ...with low excitation power. By repeating these measurements for different fluorescent proteins and by comparing...protein distribution. Therefore, photostability measurements can be performed with soluble fluorescent proteins... the experimental designs used to make these measurements and hope this information can help guide your... The second issue is that, in experiments that measure photostability, high excitation powers are often...

  24. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    ...candidate antibodies from the manufacturer and/or in the published literature. Note that the antibody being...candidate cell line (e.g. DepMap) – you’ll need to ensure that the parental cell lines of your chosen KO ...specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three...looking to label an intracellular target.   Figure 1: HCT 116 WT and SYT1 KO cells were labelled with...monoclonal antibody, ** = recombinant antibody. Figure adapted from Biddle et al., 2024.   Once an...separation between the wild-type and the KO population (Figure 1), further optimization can be performed to both...it can be a real challenge to troubleshoot RNAi failures. Potential explanations for RNAi not reducing ...

  25. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ...kDa. GFP and its homologues have a beta-barrel structure. Although the beta barrel has no strong affinity...binding sites per unit.  There are several ways to measure stickiness. In vitro, ultracentrifugation or gel...dimerization (Shaner et al., 2008 - supplementary figure C). However, these assays are qualitative. The ...Reticulum (OSER) assay (Costantini et al., 2012) measures homodimerization of FPs targeted to the endoplasmid...sequences or (ii) designed to form an inert, unstructured peptide and therefore consisted of glycines,... GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET biosensors (yellow...Golgi, whereas the mVenus-APT1 fusion does not (figure 2). In the mVenus-APT1, a lipidation motif, which...

  26. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    ...that overcomes many of those limitations (Figure 1). Figure 1: Opto-katanin induces fast, localized microtubule...specific place and time. Future studies with these CIB1-Rabs in other contexts are sure to reveal even more...cytoskeleton. They provide cells with physical structure, separate chromosomes during mitosis, support ...regions, laser ablation can damage other cellular structures, and genetic mutations are not easily reversible...roles played by microtubules, Opto-katanin will surely be useful for a wide variety of experiments. Opto-RhoGEFs... GEF to a specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized ...your cells will be ready for the spotlight.    Figure 2: Opto-RhoGEFs to control Rho GTPase activity....

  27. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    ...gleaned from the literature and engagement with researchers who study the protein or structure of interest....alleninstitute.org. A classic challenge in cell biology is making sure that what we observe through the microscope represents...case of protein tagging to elucidate cellular structures. Overexpression methods flood the cell with protein...visualize the precise location of the protein or structure of interest. Endogenous gene tagging is an ideal.... Furthermore, the quality control necessary to ensure the edited cells are behaving normally can be prohibitively...tagged are widely recognized markers of cellular structures, we provide guidance on how to make your own ... Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition of a useful coding...

  28. Hot Plasmids February 2024

    Type
    Blog Post
    ...variants:   Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy...incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, ...primary cells using peptide-assisted genome editing. Nature Biotechnology, 10.1038/s41587-023-01756-1. https...Although it quickly saw wide interest, its dimeric nature can interfere with some applications. Several labs...molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038/s41592-023...2023). A monomeric StayGold fluorescent protein. Nature Biotechnology, 1–4. https://doi.org/10.1038/s41587...observed in young macaques and vascular bias in mature marmosets. CAP-Mac also robustly expressed fluorescent...

  29. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    ...the proper controls needed to pressure-test your FRET assay to make sure it is working? This article addresses...resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific...donor/acceptor fluorophores (Stryer, 1978), a measurement that is unaffected by intervening proteins, lipid...blog has an excellent primer on FRET and how to measure it). Molecular mailbox: How to get your fluorophore... on the same street as the donor), then FRET measurements are possible. In fact, even if the majority ...properly targeted donor/acceptor pairs, then any measureable FRET will be swamped out by a sea of uncoupled..., but even these modifications can affect the structure or function of certain proteins. Rather than shove...

  30. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps of AAV production...understand how different titers are measured. A TCID50 measurement reports an infectious titer, meaning...Whitney, O., Walther, N., & Maurer, A. C. (2023). Transgene Expression in Cultured Cells Using Unpurified ...with standard molecular biology tools and tissue culture experience. Here, we’ll go over the basics of rAAV...rAAV production.  Of course, you will want to make sure that your final stocks only contain rAAV. To minimize...contamination, use appropriate decontamination procedures (Korte et al., 2021) for all disposable items...into contact with the rAAV and apply common cell culture practices diligently throughout the production ...
Showing: 151 - 180 of 1025 results