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...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...

...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...

...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...

... 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...

...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...

...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...