...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...

...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...

...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...

... 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 ...

...antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context. Sci...However, it was (and still is) infeasible to generate highly specific antibodies for every potential new target...interest with unrelated peptides that already had high quality antibodies available - aka peptide tagging...tagging.  Key to this strategy was the existence of high quality antibodies for well defined peptides. Munro...the neuropeptide substance P. This protein had a highly specific antibody to the C-terminal five amino ...sounds less than ideal.  Unexpected connections Right around the same time that Munro and Pelham were ... (1) Locate a promising monoclonal antibody with high specificity. (2) Narrow down the specific epitope...

...Every student about to graduate shares the same nightmare: the question “So, what comes next?” For those...education or ending a Master’s program, the answer might be to continue in academia and pursue a PhD. However... such as Zoom, to determine if a program is the right fit for you without the financial and logistical...to around twelve professors and heard back from eight. These emails don’t have to be long; the idea is...you a chance to get to know the faculty that you might be working with for the duration of your program...that will help you determine if the program is the right fit. Reaching out to PIs also means doing plenty...close to your computer with things you want to highlight in the interview is another way to make the virtual...

... an MRI, you know that magnetic fields can pass right through our tissues unimpeded. Andrew and Maria ...for a magnetic response among other proteins that might serve as a starting point for developing useful ...Andrew and Maria explained, “to our surprise and delight,” the very first protein they tested — E. coli ...coli expressing good ol’ EGFP — showed a slight but detectable change in fluorescence when a small magnet...team was careful to test the possibility that this might just be an artifact and eliminate sources of interference...expressed and purified. Although a ~1% change in signal might not make a very powerful tool, it was promising ...enhanced for future MagLOV-based tools, just as light-induced conformational changes are the basis of ...

... and image analysis software are highly sensitive to detecting light signals not visible to the human ... that the resolution of light microscopy is limited by the wavelength of light and, practically speaking...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...well-characterized marker proteins tagged with FPs to highlight various subcellular structures. Please see Figure...visualize subcellular structures. For example, you might use your fluoresent protein fused to a signal sequence...complexes with. These “colocalization” studies give insights about the proximity of two proteins within the...PMC3074624. 5. Bindels, Daphne S., et al. "mScarlet: a bright monomeric red fluorescent protein for cellular ...

...keep in mind that UV light only kills microorganisms that it strikes directly. UV light exposure is also ...ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half an hour can help...also harmful for eyes and skin so keep the light off when the hood is in use. Wipe the hood with a disinfectant...source of contamination for your experiments, the bright side is that you also have the power to take the...morphology: Shape and size matter You should have a light microscope close to the hood so you can view your... morphologies and doing a quick check under the light microscope will help you identify bacterial and ...are sensitive to pH. Anything between orange and light orange usually indicates that the cells have grown...

...verify loss of minicircle RNA Delivery DNA-Free High High Transgene-free and vector-free Multiple transfections... Pros Cons MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome integration Insertional...reactivation, residual expression Lentivirus Integrating High Low Efficient and Stable Genome integration Insertional...transfections required Protein Delivery DNA-Free Low High Transgene-free and vector-free. Slow and inefficient...reprogramming efficiencies ranking as low, medium or high is based on the reprogramming efficiencies reported...pMXs or pMSCV, infect dividing cells, and have a higher efficiency of generating iPSCs compared to other...integrate or replicate. Their small size leads to higher transfection efficiencies and they tend to express...

... cell or neuron type may not work well for a neighboring cell type, even within the same brain region ...Low-magnification images showing viral spread. (D-F)  High-magnification images immuno-stained against dopamine...frustrating and often months-long quests to find the right AAV for your system or to successfully replicate...sharing of negative data.  Negative AAV data is highly desirable  In a survey we conducted to improve ...and that it often provides incredibly valuable insight into one’s work. As a community, we must therefore... descriptions, a minimum n = 2, and well labeled high- and low-magnification images demonstrating vector... do you have sitting in computers and notebooks right now?, I’m willing to bet the answer would be along...

...from Addgene right on our plasmid pages and provide a citation that you can put straight into your reference...our website using the search bar found at the top right of every page. You can search for plasmids by name... number, or keyword. Although an initial search might give you many results, you can narrow your search...search results using the filters found on the right side of the search page. A great breakdown of our search...special collections pages. These pages serve to highlight plasmids that can be used for specific purposes...Every quarter we release a Newsletter where we spotlight some of the plasmids that have come through our...page - each day we showcase a new plasmid that you might otherwise miss. We hope this feature will draw attention...

...Every few months we highlight some of the new plasmids, antibodies, and viral preps in our repository...different genomic loci — in some cases, over 20-fold higher editing than PEmax. The authors hypothesized that...immunoblotted against vinculin as a loading control (right panel). Image from Addgene. These antibodies... subthreshold voltage fluctuations, while having high fidelity during action potentials of fast-spiking...j.neuron.2023.03.009. PMID: 37015225.  Simple and highly specific targeting of microglia with AAV By Alyssa...human IBA1 promoter (hIBA1a, 466 bp) that drives high microglia-specific gene expression when incorporated...hIBA1a promoters in mice. B) Quantifications showing high microglia specificity of GFP expression for the ...

...upper right) and a mixed population of B cells and dendritic cells (DCs) in the lower middle right. DCs...generated often looks like the graph on the left, it might make more sense once you realize events are plotted...plotted on an invisible Cartesian plane (right.) Each event is plotted by the amount of signal read from...cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive for...another two are B+. Our A-B+ cells are in the lower right corner.      Figure 2: This flow plot has... (SSC) and forward scatter (FSC), which measure light that “scatters” past the cell, instead of fluorescent...DCs), mDCs and pDCs. Let's follow it from left to right.  First, SSC and FSC are measured to differentiate...

...neuron-optimized viral vectors, and high-throughput screening approaches. Here, we’ll highlight these recent advancements...least, more straightforward than generating cDNA for each gene isoform individually. To highlight this advantage... The Day Lab used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA...targeted anywhere in the genome, it is relatively straightforward to use CRISPRa to selectively target these ...-optimized lentiviral vectors can be found here. High-throughput screening strategies Although the tools...

...FRET signal you should choose the highest quantum yield donor, the highest absorbing acceptor and fluorophores... similar brightness - even though in the most popular pair, CFP has five-fold less brightness than YFP...that case the cross-talk is high and your background signal may be higher than the signal generated by... the acceptor (linking the quantity of absorbed light, at a given wavelength, to the concentration of ...that can affect FRET measurements include: the brightness of a fluorophore pair, donor:acceptor stoichiometry...the donor is excited by a specific wavelength of light and the signal is collected by using emission filters... of the donor is increased. This method is straightforward and quantitative, but it is destructive and...

...Every few months we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...Improved voltage indicator: JEDI-2P Novel class of light-gated potassium channelrhodopsins Myc-tag antibody...indicator (GEVI) called JEDI-2P. Using a custom, high-throughput screening platform to apply saturating...10.1016/j.cell.2022.07.013.   Novel class of light-gated potassium channelrhodopsins by: Mike Lacy ...Xue and John Spudich identifies a novel class of light-gated potassium channelrhodopsins, named Kalium ...2022). The authors show that HcKCR1 and HcKCR2 have high selectivity for K+ over Na+ and rapid photocurrent... optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na+ and other cations...