We narrowed to 1,049 results for: URE

...without DNA cleavage. Nature, 551(7681), 464–471. https://doi.org/10.1038/nature24644 Gaudelli, N. M., Lam...double-stranded DNA cleavage. Nature, 533(7603), 420–424. https://doi.org/10.1038/nature17946 Komor, A. C., Zhao,...other or one pyrimidine base to the other. Figure 1: Base transition editors catalyze purine-to-purine...strand as a template, copying the base edit. Figure 2: Cytidine deamination takes place on the free...base editing efficiency for mammalian edits is to ensure the editors make it into the nucleus and that they...and specific CRISPR-based cytosine base editing. Nature Biotechnology, 41(5), 663–672. https://doi.org/...increased activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https://doi.org/...

... 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...

...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...

...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 ...

...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....

...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...

...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...

...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...

...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...

...introduced mutations that drive leukemia (Figure 3). Figure 3: Using LARRYv2 libraries for state-...expansion cultures. A) Experimental design. B) UMAP of cell states in ex vivo expansion cultures from days...maintaining high brightness and fast maturation time (Figure 1). See the paper for more details on small differences...mGold2t depending on the application. Figure 1: Super-resolution imaging of actin filaments ...yellow fluorescent proteins for extended imaging. Nature Communications, 16(1), 3241. https://doi.org/10.1038... an RNA guide (known as an omega RNA, or ωRNA) (Figure 2), but TnpB is small enough to be carried by viral...less than a third of the size of Cas9). Figure 2: Comparison of TnpB (A) and Cas9 (B) genome editors...

...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 ...

... efficiencies using sequence features and DNA repair determinants. Nature Biotechnology 2023, 1–11. https... evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants...Once we had our datasets with all the different features and editing efficiencies, we could train our prediction...very diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency...downstream flanking sequences to their input string (Figure 2). This string can then be used as input for the...e.g., top 5) and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with...Mathis et al., 2024) and concurrent research by the Shendure lab (Li et al., 2024) have highlighted how chromatin...

... SDS-PAGE, measure the protein content and normalize to ensure equivalent loading. Denature the samples...antibodies.   Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated...denaturing western, the protein is denatured to its primary structure and separated by size with smaller...western, the protein’s secondary and tertiary structures remain intact and the protein is separated through... expensive system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack...its native state while a western blot detects denatured polypeptide chains. The protein regions available... antibody validation. This level of validation ensures that the antibody is staining the intended target...

...of Addgene’s work culture. Our developers are a diverse group of ethnicities, cultures, genders, educational... for working on a new feature. We start with a story that describes the feature and what the criteria ...stakeholder accepts the new feature as done and we’re onto the next story. Figure 1: Addgene’s Agile Approach...working on a feature, sometimes we have to take short cuts or add a band-aid to get a feature to work. We...number of stories we commit to for each sprint, to ensure we will have the capacity to finish all of the ...criteria is for accepting the feature as done correctly. These initial stories are usually a result of collaborations...step-by-step instructions on how to demonstrate the feature. For example, we recently reduced the number of...

...sequence (Zong et al., 2018) (see CBE-3 in Figure 3). Figure 3: Activity windows of plant base editors...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)... that compete with the desired outcome are not pictured. Adenine base editors (ABE) What if you have...satisfactorily accepts DNA as its substrate (see figure 2).          The artificially evolved adenosine...genomic A-T base pair is converted to a G-C pair (Figure 1). As the cellular inosine excision repair is ...base editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular...

...better at 30°C or room-temperature, which is accomplished by growing your culture to the desired density...previous Plasmids 101 blog, we reviewed the salient features of several popular strains of E. coli for DNA ...except ** which are K12 Strain Resistance Key Features Genotype Use BL21 (DE3)   Basic IPTG-inducible...Denotes the presence of an additional plasmid-- make sure to maintain this by growing on appropriate media...timing is important, as you typically want to make sure your cells have first reached an appropriate density...expression. If you wait too long to induce, your culture will start collecting dead cells, and, conversely...too early as there are not enough cells in the culture to make protein.  The DE3 lysogen/T7 promoter combination...

...acceptor fluorophore can be measured using microscopy techniques. FRET measurement sensitivity makes it suitable...one of the first techniques which enabled the measurement of single molecule interactions in living cells...living cells. Since then FRET has also been used to measure conformational changes, cleavage activity using...is still one of the best and most used pairs to measure FRET. The table below lists plasmids that can be...and Venus Other issues that can affect FRET measurements include: the brightness of a fluorophore pair...between FRET efficiency and cross-talk. Methods to Measure FRET for Cell Biology Studies Several methods have... have been used over the past 20 years to measure FRET and there is not one that is better than another...

...environments: corrosive pH ranges, crushing pressures, near-melting temperatures, tons of radiation, or freezing ...Sreepadmanabh M from IISER Bhopal. All illustrations / figures featured were designed by our team member Kallol Mozumdar... take bacteria out of their optimally suited temperature ranges and make them grow better at suboptimal...cold-loving) bacteria. These bacteria inhabit cold temperature locales and psychrophilic enzymes help them survive...next-door microbes which grow optimally in moderate temperatures (usually defined between a fairly nippy 20°C...materials degrade at a much slower rate under cold temperatures, and frigid conditions decrease the potential...mesophilic host systems. The so-called “regular” temperatures of growth, say around 37°C, is a far cry from...

... new generation of deaminases. Figure 2: Structure of ABE8e*A showing dimerized TadA subunits... need for assays that capture multiple layers of editor behavior. Figure 3: Labeling of Cas9-mediated...never been shorter. As cryo-EM structures, molecular simulations, and structure informed diffusion models ...influence both efficiency and fidelity. Figure 1: The first steps of prime editing. DNA release...guide‑directed off‑target workflows often fail to capture newly described editing outcomes or meet emerging.... Chicago) presented Inrich-seq, a method that captures the ABE intermediate inosine directly in cellulo... presented PERT: prime editing readthrough of premature termination codons (Pierce et al., 2025). The ...

...method does provide a quantitative measure of autophagy, it only measures bulk autophagy (as opposed to giving... Long-lived protein degradation: This approach measures the degradation rate of radio-labeled long-lived...long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for several hours...by measuring the amount of radioactivity in the culture supernatant (this is indicative of protein degradation...out of this assay. This is also a quantitative measure of autophagy, but, like the protein degradation...degradation assay above, it’s slow and only measures bulk autophagy. Morphological assays Electron microscopy...method relies on the identification of autophagic structures based on morphology. Autophagosomes are relatively...