We narrowed to 856 results for: IGH@

...Every quarter we highlight a subset of the new plasmids in the repository through our hot plasmids articles...articles. These brief articles highlight the main features and applications of a partiular plasmid or ...parallel analysis that successfully identified new, highly specific agonists for orphan GPCRs demonstrating...are found in one third of all cancers, including a high percentage (up to 95%) of pancreatic cancers. Ras...an additional 6 gRNAs per gene generated using slightly relaxed gRNA design parameters. To demonstrate...method in bacteria that enables rapid, automated, and high-throughput genome editing is multiplex automated...otherwise unmodified bacterial strains (5). MutL is highly conserved in distant relatives of E. coli so it...

...sometimes these fluorescent proteins might not localize to the right places, which can provide misleading...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...efficiency of tobacco etch virus protease (TEV), a highly sequence-specific protease used to cleave affinity...work for evolution of other target proteins and might be useful for your studies.  Find the plasmids to...including fusions with Histone 2A or 2B can result in bright targeted expression while minimizing non-specific...microtubules of mammalian cells, the author found that too high expression of these markers can lead to non-specific...biotin and biotin-4-fluorescein to be dissolved at a higher concentration, and results in uniform NB biotinylation...

...lowest-ranking active (green) variant but with higher activity than the highest-ranking inactive (red) variant from...nuclease variant cleaves a target, all variants with higher activity will cleave it too. This leads to the ...variant which still retains on-target activity (highlighted by a bold frame) provides off-target–free editing...low-activity variants (red rectangles). However, among the higher-activity nucleases, the weakest variant that is...on-target prediction tools to increase the likelihood of high efficiency cleavage. In our experience, DeepWT provides...Identify the least active variant that still shows the right amount of on-target editing. This variant will ...active variants with the lowest activity, and thus highest fidelity, in the full set). Adapted from Kulcsár...

...Addgene, we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...strategy to fix the issue. I was problem solving!  You might think that being able to figure out what went wrong...skills” and look at how scientists can develop and highlight these valuable skills. Hopefully, having a better...everything or revealed another issue in the process, you might need to go back to the beginning, reassess the situation...is just a question you have about the world. You might do some background research to better understand...issue. Depending on the particular problem, you might even be expected to write up a report to communicate...

...using a highly competent strain that you can use for long-term library storage. After overnight growth,...popular classes of phage proteins to study. These are highly useful for the biotech industry, which is in the...the plate so you can regrow the lifted colonies overnight), flip the membrane over and place it colony-side...tested, as with any protein purification, but overnight at 30°C is a good start. Prepare a lysis solution...repeated 5 times) works well. Leaving the membrane overnight in the freezer during one of these steps is also...with your cells of interest and growing them up overnight. Assuming they expressed well, the proteins encoded... it can really help to incubate the membranes overnight in 500 mM to 1 M NaCl in order to enhance folding...

...color: they strongly absorb visible light to give colors in ambient light. In contrast, the widely used green...protein (GFP) gives a bright green fluorescence when exposed to blue to ultraviolet light, whereas chromoproteins...chromoproteins absorb visible light and give off color in ambient light, it gives scientists the ability...cases, cells containing high-copy plasmid-borne chromoproteins lost color overnight in liquid culture, though...colorful corals, to name a few. What’s behind these sights are an abundance of biological molecules like chromoproteins...Cassiopea xamachana (Blanquet and Phelan, 1987). Eight years later, scientists isolated pink, purple, and... irradiation by reflecting visible and infrared light to dissipate excess energy at wavelengths of low...

...substantially brighter and more photostable than fluorescent proteins, providing a straightforward way to improve...refinements to these systems—smaller tags, brighter conjugates, higher fluorogenicity, photoactivation etc.—...fluorophores conjugated to molecular species with high affinity for endogenous molecular targets such as...selectivity of the fluorophore attachment, the brightness of the resulting conjugate, and the complexity...exhibits low fluorescence when free in solution, but high fluorescence upon binding to its cognate protein...due to its established chemistry, small size, brightness, and cell permeability. In past decades, further... the Alexa Fluor and ATTO dyes, with improved brightness, photostability, and spectral range. Nevertheless...

.... The iBAR method resulted in high quality data even when used at a high multiplicity of infection, because...also function in high salt conditions - up to 100 mM NaCl. These tTALE-FPs also have higher DNA binding affinity...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...next generation sequencing.  In order to avoid a high false-positive rate discovery rate, conventional...human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low number of cells... levels within the cell. When levels of Ca2+ are high in the cell, calcineurin dephosphorylates the protein... the bacterial βgal assay, and are not suited to high throughput screening, calcineurin dynamics measurements...

... Zdk1 binds LOV2 with high affinity in the dark but dissociate under blue light. This tool has now been...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...up here.   Listen this episode of Hot Plasmids! Light controls the activity of a microtubule-associated... lab at UCSF to modulate cellular dynamics with light by modifying EB1. EB1 normally sits on microtubules...microtubule growth can be switched off with blue light which causes LOV2 and Zdk1 to dissociate due to ...photodissociable EB1 could be stopped in areas with blue light - a finding which opens novel ways in manipulating...Scientists are always on the lookout for better, brighter fluorescent proteins (FP) with improved tissue...

...development. However, careers outside of the lab highlight “transferable” skills such as leadership, management... impact, or a journalist does not understand the high-level science discussed in an interview, your project...be taboo in the lab, multiple research studies highlight the importance of taking a break and incorporating...network Attend informational interviews to gain insight into the daily routine of the potential job Join...and research 2 – 3 potential professions will highlight which skills you should focus on improving and... skill sets are strengthened. This short list highlights the ubiquity of skills across professions within... Outreach activities (e.g. Volunteer at a local high school science class: Science Club for Girls) ...

...Save time, go high throughput You are ready to perform your assemblies, but you have a high number to get...E. coli strains with higher transformation efficiency. If you’re cloning in high-throughput, we recommend...ligation efficiency, we recommend you calculate the right volume of each plasmid according to their concentration...time to think big and perform your assemblies in a high-throughput format. If your lab owns a liquid handling...restriction–ligation reactions in a 96-well (or higher!) format. Liquid handling robots are not only faster...experimental costs. If you are planning to perform a high number of assemblies, you will also likely need ...your bacterial stocks beforehand. You can also use high-throughput methods to streamline your plasmid extractions...

...cell is artificially high for both plasmids, so each plasmid “thinks” it has a higher copy number than it... 101 post, we’ll cover why this happens, how it might affect your work, and how understanding it can be...inhibits replication when copy number in the cell is high, but allows replication when copy number in the ...replication machinery when plasmid copy number is high (del Solar et al., 1998).  In cases of compatible...plasmids, though the partitioning problem does occur in high copy plasmids (Diaz et al., 2015). Plasmids contain... plasmid pairs into the new daughter cells.  For high copy plasmids, incompatibility due to partitioning...incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like binding...

...amino acids (see example to the right). With such simple rules, you might think it’s easy to come up with... entire translation process. For example, during high growth rate conditions, E. coli preferentially upregulates...production of tRNAs that recognize codons found in highly expressed genes (Emilsson and Kurland, 1990). Controlling...produced at a lower rate than proteins encoded by highly abundant, charged tRNAs. For example Tuller et ... protein is encoded by a mixture of codons with highly and poorly charged tRNAs, different regions of ...usage, an organism can change which proteins are highly expressed and which are poorly expressed by producing... during amino acid starvation thus resulting in higher production of amino acid biosynthetic enzymes (...

...aggregation tendency, increase its brightness, and decrease the amount of light required for dimerization. She...Fluorescent Proteins Use Light to Control CRISPR Genome Editing Choose the Brightest Fluorescent Protein for...future award opportunities. Xin Zhou: Fluorescent Light-inducible Proteins (FLiPs) Fluorescent proteins ...create enzymes that can be turned on and off with light.  Xin hypothesized that she could make enzymes controllable...known to oligomerize when exposed to ultraviolet light. To do so, she generated fusion proteins in which...enzyme’s active site. In these fusions, ultraviolet light exposure caused oligomerization of the Dronpa domains...shutting off the enzyme. Later exposure to cyan light stopped Dronpa oligomerization and turned the enzymes...

...some light on multicolor imaging with fluorescent proteins. With the right microscope and the right choice...when choosing an individual fluorescent protein (brightness, photostability, and so on; see the previous ...colors and the mCherry spectrum is shown in the lighter colors; excitation spectra are blue and emission...this filter set, we would expect TagRFP to give a brighter signal than mCherry. In general, filter sets designed...supplement your cells with biliverdin for maximal brightness. In mammalian cells, one of the improved folding...fluorescent protein that is supposed to be extremely bright. In S. cerevisiae, we've tested a number of green...proteins with this filter set and have reported brightness measurements. Here, EGFP outperforms the improved...

...are commonly used in the lab as tools and we all might benefit from getting to know them a little better... injury, retroviruses generally have moderate to high immunogenicity, meaning they induce an immune response...transduce (infect) a wide range of cell types with a high efficiency. However, gamma-retrovirus infection ...tools have been derived from. Lentiviruses have slightly larger packaging capacities than gamma-retrovirus...immunogenicity, meaning it does not induce the same high immune response as retroviruses. This virus can ...been identified so far! Adenovirus generally has high immunogenicity, but some strains have been engineered... discussed in this article, adenovirus has the highest maximum titer, a plus for production and infection...

... university. My to-do list might look something like the image on the right (and for those of you who ...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...tasks at the expense of time spent on the long and highly useful tasks. The quick accomplishments will likely...you the energy to finish any additional work you might need to do. In most cases, I would recommend the...constantly switching between different projects, you might not be able to make any reasonable progress on any...to-do lists at the end of the day so they can jump straight into tasks the next day. If you work in a job ...

...ideal characteristics (low aggregation, high photostability, and high stability after fixation.) From the ...still being developed today. In this post, I will highlight the evolution of the  Brainbow multicolor labeling...zebrafish and Drosophila. Why Brainbow? Brainbow highlights the trend in biology towards single-cell resolution...development of a new technique. In a structure as tightly packed and interwoven as the brain, robust single-cell..., one would expect ten colors (see table to the right); in various reports, Sanes and Lichtman have observed...observed from 90-160 distinct colors due to higher copy number. The name Brainbow is truly fitting for...fluorescently distinct, their protein sequences are highly homologous, preventing design of antibodies specific...

...bivalent, so may have higher avidity than monovalent Fab’ or Fab fragments. Higher avidity means that F...’ll go over what a Fab fragment is and when you might use it in the lab. What is a Fab Fragment? Consider...chains (two identical heavy chains and two identical light chains) each with a constant region and a variable...heavy chains and between one heavy chain and one light chain. When assembled, the four chains make a Y-...are the “arms” of the Y and comprise the entire light chain and a variable and constant region of the ...important features labeled. V = Variable domain. L = Light chain. C = Constant domain. H = Heavy chain. Created...function but not the Fc region (more on when that might be later). Find scFv plasmids at Addgene Depending...

...the contents. Tightly coupling barcodes and containers might not be such a bad idea, tightly coupling them...we’ll highlight some of the lessons we’ve learned over the years with the hope that our insights can help... help steer you in the right direction when writing your own software. None of this is gospel, but we ...help tell this story. In this first example to the right (# this will make you sad),  someone has put in ...numbers. Loosely couple containers and contents Right from the get-go be as clear as you can be (there...them to their contents might leave you sad and confused when a tricky procedure failed and one of your...In the first example we’ve used inheritance to tightly couple the contents of a tube with the tube itself...

...only scientists with an MSc or higher apply. It was important to weigh the cost to benefit of studying... I didn’t need to explain why I changed jobs. I highly recommend taking on a similar position in your ...your field of interest before you dive straight into a full-time position. For my part, I really enjoyed ...for a disease that traditional medicine couldn’t fight effectively. I had to learn how to cope with a different... companies which are rapidly expanding, as this might not be sustainable. After working at this company... of different types of pancreatic cancer and highlighting which treatment would produce the best outcome... does not work for all patients, leading to very high mortality. I really loved the fact that my work ...

...error — both human and biological. Even the most high-fidelity polymerases can make mistakes and lead ...these errors. These processes allow us to have a high standard of sequence quality across all plasmids... same QC process to ensure all plasmids pass our high-quality standards and contain the expected plasmid...accurate The gene/insert or any major feature highlighted by the deposited scientist  The backbone elements...deposited to Addgene. This review process also highlights other QC topics that may require additional attention...Illumina MiSeq platform, such as GC-rich regions and highly repetitive sequences and/or features. Most of these...assembly in Geneious can still provide additional insights to resolve the assembly and determine if there...

...easily detect and measure the light they emit. Proteins that give off light include chemiluminescent proteins...victoria there is a ring that glows with a faint green light. To investigate this green glimmer, Shimomura collected...which he called aequorin, gives off a faint blue light when it binds calcium ions, and the second, which...which we now call GFP, absorbs that blue light and glows green. In the late 1980s, another researcher, Douglas...protein localization. And as it turns out, he was right! He began to study the A. victoria gene responsible... fell into place, as they, taken alongside the insights provided by the studies in Tsien lab, covered ...their work on GFP. This great honor serves to highlight how useful fluorescent proteins like GFP really...

... and Mark Brigham have taken the technology to another level with the addition of two light-sensitive ...occurs within minutes of light exposure. Third, the response can be graded with light dose and is fully reversible...reversible after light retraction. Finally, because light can be applied non-invasively, its use is not...borrowed from Arabidopsis thaliana. The results are light-inducible transcriptional effectors (LITEs) designed...them on or off, literally at the flip of a (blue) light switch. Zhang’s team devised the plasmids now available... transcription in as little as 30 minutes after light stimulation. Under those optogenetic controls, gene...several advantages of the new system: "First, the light-responsive molecules of cryptochrome 2 are the chromophores...

...understand how chromatin might influence editing results at your target. Figure 3: High-throughput screening...comes in! Evaluate prime editing efficiencies in high-throughput libraries My labmates and I wanted to...of pegRNA designs based on patterns observed from high-throughput experiments, where we can evaluate tens...of designs in parallel. To do this, we performed high-throughput ‘self-targeting’ screens with oligo libraries... edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and building...screens such as PRIDICT/PRIDICT2.0 provide valuable insights into pegRNA editing efficiencies for specific ...research by the Shendure lab (Li et al., 2024) have highlighted how chromatin substantially affects prime editing...

...needed, without a high number of samples (~5). Tukey’s test – Most conservative test. Higher false negatives...select the right test for the job (and, uh, gentle reminder: keep your friendly neighborhood biostatistician... can tell you, and how to identify which one is right for you. Statistics – The basics Statistical analysis...ANOVA. Isn’t that what a two-way ANOVA test is you might ask? Not exactly! A MANOVA test assesses the effect...could test the impact of temperature, humidity, sunlight exposure time, and watering volume on total crop...total), your likelihood of a false positive is much higher than if you only look at 2 or 3 pairwise comparisons...range test – Most powerful. Low false negatives, high false positives. Control vs. all samples Oftentimes...

...even different chimeras might function better. In that case, gene shuffling might be a good option. In gene...random library of mutants using error-prone PCR might be a good solution. The process of error-prone PCR...you wouldn’t want errors in a PCR and would use a high-fidelity polymerase to ensure accuracy. In this ...more control, structure guided directed evolution might be more your style. Instead of leaving sequence ...regions you want to evolve. With this added layer of insight, you can shrink your mutant library and increase... of directed evolution, all while maintaining a higher degree of control and efficiency! Screening mutant...improved properties across large mutant libraries, high-throughput screening techniques such as ELISA and...

... colony formation on agar plates, less stable at high temperatures and in acidic environments.   Carbenicillin...better’ antibiotic than amp: it is more stable in high pH environments and is less susceptible to degradation..."fontawesome-5.14.0" name="Check" style="SOLID" height="16" purpose="decorative" title="Check icon" %}...

...common reagents, straightforward protocols, and standard microscopes sounds great, right? But there is always...         Why Expansion Microscopy?  Traditional light microscopes are limited in the level of resolution...everything needed for synaptic transmission, are right around 250 nm, making it difficult to study the ...the inner workings of the synapse by conventional light or fluorescence microscopy (Dani, et al., 2010)....What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem. Instead of imaging..., biological samples are enmeshed in a gel of a highly absorbent material that is able to expand evenly...points with a conventional, diffraction-limited light microscope.  Along with using more accessible imaging...

... the assayed PPI. This high-throughput application is much more straightforward to carry out because it... This is straightforward in easy-to-transfect cell lines where plasmid dosage can be tightly controlled...the non-radiative transfer for energy between neighboring fluorophores, FRET can be used to detect the ...subcellular localization of the studied PIPs. The straightforward nature of BiFC also makes it easier to generate...generate a large number of constructs for high-throughput screening of PPIs. For example, Bischof et al....Hu, 2012). Having a rough idea of how your PIPs might interact will allow you to determine where to fuse... Proteins 101: GFP Fusion Proteins - Making the Right Connection. Cellular expression Whether BiFC constructs...