...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...

...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...

...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 ...

...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...

... 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...

... 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...

...protein at higher levels, you risk observing artifacts only present when the protein is at a very high concentration...membrane-targeted CAAX-SunTag was 18-fold brighter than sfGFP! The high SunTag signal allowed them to cut their...transcription factor binding sites in a promoter lead to higher transcriptional activation. Multiple nuclear localization...explosion SUperNova," SunTag can help you turn up the brightness in your fluorescent imaging experiments. Fluorescent...their laser power by over 80% and still obtain a higher signal-to-noise ratio with a lower photobleaching...mitoNEET-SunTag-GFP. As expected, they obtained bright images of mitochondria without organelle toxicity...to traditional fluorescent proteins Improved brightness and signal-to-noise ratio Less chance of phototoxicity...

...elevated plus maze, the light-dark box is a simple device comprised of a brightly lit (risky) and a dark... a camera or tracking software. Locomotion is a highly complex process on the level of individual neurons...findings by controlling neurons in the mPFC with light. Activation of these neurons via the channel Channelrhodopsin...inhibitory mechanism of locomotor sensitization and might open new ways for a novel therapeutic approach against...to stay in dark areas (protected) as opposed to brightly illuminated ones (risky). They also prefer to ...diseases, animals have reduced anxiety levels and a higher tendency for risk taking. These animals also show... arm (risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open...

...make sure people pay attention. Highlight your lowest points and highest points and make clear what was...I’m working with a talented journalist to shine light on living as a scientist in Beijing, talking to ...judgement. Eventually, we published the paper in a high-impact journal and it worked out for all of us.”...cases, the protagonist will be you. Make sure to highlight what lessons you’ve learned and what knowledge... into your research at an outreach event as you might in a scientific paper. Build lots of versions of...difficult and what was easy when appropriate. It might not be scientific to bring emotional discovery into...people pay attention. Practice Nobody gets good overnight. The only way to improve is to keep working on...

... is the cr:tracrRNA approach highly efficient, it is also straightforward as the RNA molecules can be ...events. In the light of these results, we re-analyzed our data and found that 75% of our highest knock-in efficiency...of interest are pulled-down and later coupled to high-throughput sequencing to identify genome-wide binding...Telmer 1998). Indeed, this method can be used at high throughput because numerous cell lines can be generated...challenges, we set out to develop a hassle-free, highly efficient Cas9 protein-based method for epitope...which, in our hands, is both cost-effective and highly active. After csRNP complex formation, a single-stranded...transfection/delivery step needs to be optimized to achieve high knock-in efficiency. To calculate the tagging efficiency...

...dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright, & Ebright, 2001). These Cy/NTA conjugates work the ...6. Kapanidis, Achillefs N., Yon W. Ebright, and Richard H. Ebright. "Site-specific incorporation of fluorescent... James D. Fessenden, an Assistant Professor at Brigham and Women’s Hospital. Biochemists often struggle...mailbox: How to get your fluorophore delivered to the right place? FRET experiments require targeting the donor...the proper mailbox (i.e., a binding site) in the right city (protein). In a cell, this task is even harder...acceptor molecules are targeted nearby (such as a neighboring house on the same street as the donor), then ...specificity Donor fluorophores need to go to the right place. Mis-targeted donors never “see” FRET acceptors...

...aberrant light chain sequence, but was predicted to occur at a low frequency within functional light chain...a degenerate primer set to amplify IgG variable light (VL) and variable heavy (VH) chain sequences. Fusion...plasmid backbone for expression of the mAb heavy and light chain, each from a constitutively active CMV promoter... expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two...express the target antigen could be screened in a high-throughput manner. Cells were labeled with both ... R-mAbs. It was recognized that an aberrant IgG light chain transcript was expressed by some of the hybridomas...cloning, which in most cases left the functional light chain intact, the authors were able to greatly increase...

...Addgenies support a high collaboration, high communication, high efficiency, high quality, low meeting...getting ready to embark on new exciting projects. Right now we don’t have to worry about succeeding at our...materials to better serve the community. We are delighted at the early outcomes and the ecstatic reception...by the scientists who request from us but also by high school students and industry scientists as a universal...21,806th most popular website in the world. I know, right? Upgrades to the internal LIMS pages and excellent...excitement of what we are accomplishing and what more we might do. I am grateful every day for their dedication...viral_sharing=False, embed_button=False, width='1280', height='720', player_id='7039679868', style='' %} Resources...

...two recombinases. How might one read the state of an RSM? The most straightforward way to read the state...state machines in living cells” (1), into a more straight-forward, concise explanation of how to adapt our...exposition, and detail, please refer to the paper. Why might one be interested in state machine technology? Biological...sent the PCR product for sequencing.  A less straightforward, but more population-wide (as opposed to single-cell...method since it lends itself to cheap, quick, and high throughput state detection with flow cytometry. ...cells; the sequencing and qPCR methods do not. How might one implement RSMs? In our study (1), we implement...Figure 3 below): Input Plasmid: The input plasmid (high copy, ColE1 origin of replication), contains large...

... recommended.  Environmental influences like bright light or noise should be avoided as this will also...consideration for behavioral testing in a laboratory, a high level of experimental control is required, because...frequencies (> 20 kHz, produced from fluorescent light bulbs, for example) that are outside the humans’...optogentics to control neuronal activity, commonly via light-activated ion channels. Similarly, chemogenetic ... trigger neuronal excitation or inhibition upon light (or drug) application.  Genetic manipulation can... generally mediates broad cell tropism and has a high percentage of neuronal targeting after stereotaxic....  Viral vector injection Viral vectors allow a tight spatial and temporal control of gene expression ...

...concentrations of IL-3. Cells grown in high concentrations of IL-3 show a high proportion of blue fluorescence...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...Split-wrmScarlet is a new split red FP that is three times brighter in worms than split-sfCherry3. It was created ...in live cells. The fluorescent timer emits blue light around the time of synthesis and, after a few hours...cells. Cells undergoing rapid cell cycles have a high blue to red ratio, whereas cells with long or slow...cells as measured by flow cytometry. Cells grown in high IL-3 showed mostly blue fluorescence, indicating...CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids. To find all of the...

...Atsushi Miyawaki and colleagues identified a highly bright and stable fluorescent protein, StayGold. Although...Every few months we highlight some of the new plasmids, antibodies, and viral preps in the repository...uses a much shorter crRNA. Cas12-RNP-PAGE achieved high editing frequency (nearly 100% in some cases) and...break up the dimerization without losing that brightness and stability. Here’s a quick overview of these.... This variant has similar photostability and brightness compared to the original, though its maturation...sufficient to disrupt dimerization while maintaining the bright and stable fluorescence of the original (Ivorra-Molla....rs-2684100/v1.  Piatkevich, K., et al. (2023). Bright and stable monomeric fluorescent proteins derived...