... a DOI referencing primary literature, and every effort is made to moderate and cross-check all data with...affected by environmental conditions (such as temperature, pH, fusion protein, etc...).  There are many... Addgene blog – outlining various important considerations when choosing a FP, but much of the primary... making such a decision remains spread across literature in publications that introduce these tools. ..., and I collaborated with him on a web-based interactive visualization of FP attributes (which turned ...that allows users to see when a given page has unmoderated changes and to view previous versions.  Currently... with published literature as soon as possible after submission. Using FPbase The most obvious use case...

...your incubation temperature. Some proteins aggregate at 95 °C, a common lysis temperature, so consider a...background noise. Temperature and time You may need to optimize your antibody incubation temperature and time.... variants Check literature Posttranslational modifications Check literature Highly charged...said, normalizing to total protein expression is preferable, and more accurate, than normalizing to ubiquitously...recombinant multiclonal antibodies to increase overall signal from the primary antibody.  Pro tip! If ...common conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies... including trying longer incubations at both temperatures if needed. Washes Check your antibody manufacturer...

...many distinct CRISPR therapies to match these different mutations. For each therapy, effectiveness and ...’s size makes it difficult to deliver via gene therapy, so researchers have set their sights on another...work with dystrophin exon skipping? For such a therapy to be successful, multiple requirements must be...minimal risk of off-target editing. In order for the therapy to persist over time, stem cell editing would be...in DMD, and it’s estimated that exon skipping therapies would be applicable to 80% of DMD patients. ...characterizes additional, unique facets of CRISPR DMD therapy. Long et al. show that AAV-CRISPR does not cause...represent a great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler...

...coming of age. Molecular Therapy : the Journal of the American Society of Gene Therapy, 17(8), 1316–1332. https...can be an advantage for researchers looking to generate long-lasting changes in cells. However, for some...plasmids. For simplicity, we’ll use the second-generation system as an example.  The lentiviral transfer...integration, two types of circular episomes can be generated through either homologous recombination or non-homologous...integrase-deficient lentiviral vectors? There are several reasons why you might choose an IDLV over a lentiviral...clinical settings, as evidenced by retroviral gene therapy trials for X-linked severe combined immunodeficiency... 2000s. These trials unfortunately resulted in several patients developing T-cell acute lymphoblastic ...

...biotinylation, the discovery of the biotin-avidin interaction and the uses of biotinylation in molecular biology... and energy transduction. Biotinylation is the general process by which biotin is added to a protein or...protein (BCCP). Discovery of the biotin-avidin interaction   Figure 1: Biotin is enriched in egg ... of the strongest noncovalent protein-ligand interactions found in nature. Once the bond is formed it ...it is unaffected by changes in pH, temperature or organic solvents. Uses of biotin in molecular biology...scientists began to exploit biotin and its strong interaction with avidin for molecular biology and biotechnology...that are coated with avidin. The biotin-avidin interaction has also been adapted for use in immunohistochemistry...

...we’ll go through some of the most important considerations to make when planning and performing an IF ...IF experiment. What is immunofluorescence? Generally speaking, IF is an application in which antibodies...iBiology). IHC and ICC are essentially just more general terms that can also refer to assays using non-fluorescent... visualize their target. These antibodies are generated against a specific antigen and then conjugated... more common is that secondary antibodies are generally polyclonal, which means that multiple secondary...amplification. Another reason is cost effectiveness. Generating and validating primary antibodies is a time consuming...antibodies that exist and you can begin to see why generating fluorophore-conjugated versions for each primary...

...induced binding. The lab used this general design scheme to generate two more OptoNBs, one against mCherry...loop 1 always generated OptoNBs with dark-induced binding, but insertion into loop 6 generated both blue ...purification. After binding YFP to the column, you wash several times to remove non-specific proteins, and then...on the key components of OptoNBs, how they are generated, and their use for protein purification and controlling...chain antibody domain.  How do these components interact? When fused together to form an OptoNB, the AsLOV2...nanobody’s ability to bind its target protein.  Generating OptoNBs The opening and closing of the AsLOV2...over-activated in cancer due to its involved in cell proliferation and differentiation.  Since Ras is a membrane...

...Brookline Interactive. Learn more about Carl Paratore, MBA, CPA Mr. Paratore serves as the General Auditor...Suffolk University Law School and Paratore is the General Auditor at Tufts Health Plan. The addition of Katz...perspective, what do you think are important considerations for the future of open science? What about ...University Law School. Suffolk University graduates on average 20 PhD’s a year and we have a very dominant presence...presence in the biotech sector in both patent law and general life science regulatory law. My role at Suffolk...work at Tufts Health Plan and my role there is general auditor. I oversee a department of 20 individuals...the efficiency and effectiveness of the Plan's operations. We’re looking for those “what could go wrong...

... used to generate cDNA, which  was used as a template for PCR amplification with a degenerate primer set...limitation of hybridoma generated mAbs is that if the frozen cell line becomes non-recoverable, the stock of the... distributed by Addgene.  The plasmids used to generate these antibodies were based off of a large collection... they were also able to isolate RNA from non-recoverable hybridoma stocks, without having to grow up the... the cells. This RNA could then be used to generate functional recombinant monoclonal antibody (R-mAb)...manner. Cells were labeled with both a hybridoma generated mAb and the R-mAb, with the R-mAb being verified...encoding sequence within the plasmid backbone to generate R-mAbs of different subclasses, including subclasses...

...virus. In general, viral envelopes dictate infection of target cells through their interactions with target... between neurons are what define how the brain operates, and thus, are a major part of the answer to many...individual neurons they infect. However, they generally are not transmitted to adjacent neurons via synapses...monosynaptic tracing was used to examine how adult-generated neurons integrated into the brain over time (Deshpande...This study addressed the question of how newly generated neurons integrate into the complex pre-existing...the functions of these two neuronal populations. Overall, this study laid the foundation for how different...the brain receive inputs and compute outputs. In general, RABV monosynaptic tracing is a powerful tool that...

...made much easier if the interaction between agent and customer is cooperative rather than combatitive.... daydreamed about the ideal customer service interaction - one that gives our customers the most utility...apply to any biology-related customer support interaction. Before contacting customer service In an age...that for you” meme is infamous enough to have generated it’s own tongue-in-cheek website.  If you are ...catalog number ready. I've attempted to answer several emails that start: “Hi! We ordered something from...concern. Please be courteous. We understand how exasperating it can be to lose time and money on a potentially...re going to get. There are questions that are answerable in minutes and questions that could take weeks...

...familiar with before working with mouse models, several common techniques used to create engineered mouse...are then injected into a blastocyst. There are several types of genetically engineered mice: transgenic...gene construct inserted at a specific site. To generate a transgenic mouse, microinjection of DNA or infection...mouse genome. In contrast, knockin strains are generated via homologous recombination. A targeting vector...CRISPR/Cas9-based systems have been developed for generating knockin mice but because CRISPR/Cas9 engineering...However, CRISPR/Cas9 systems are more useful for generating knockout mice. Knockout mice Knockout mice have...during the embryonic stem cell stage using one of several techniques. The longest standing technique is the...

...indicator ONE-GO biosensors kit Antibodies for neurodegeneration research   Optimized prime editing in dicots...PE efficiency barrier in tomatoes" by combining several approaches (Vu et al., 2024). Their optimized strategy...applicable to dicot species. In addition, they generated offspring tomato plants that inherited the genomic...single-molecule fluorescence techniques. After generating a series of single- and double-tagged constructs...kinesins KIF5A and KIF5B as well as backbones for generating other SNAPf-, CLIPf-, and CLAP-tagged proteins... a YFP-tagged G protein and a NanoLuc® (Nluc) Luciferase-fused detector that specifically binds to active....01.028. New recombinant antibodies for neurodegeneration research   By Ashley Waldron We recently added...

...interrogate immune cells to improve immunotherapy in glioblastoma, and generate programmable screening platforms...vectors: The Rubik’s cube of human gene therapy. Molecular Therapy, 30(12), 3515–3541. https://doi.org/10.1016... vector for gene therapy of human diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org... methods — in vivo, in vitro, and ex vivo. The general process of these screens is the same: Deliver ...interest (positive or negative selection). Next-generation sequencing (NGS) to identify genes of interest...your library for each experiment. Additional considerations One of the downsides to using AAV is the limited...Cas12a (Cpf1). Even more creative options involve generating strains of mice that stably express Cas effectors...

... strongest interaction between the guide and the target and 0 being the weakest interaction due to mismatches...such that weak interactions (e.g., scores <20) are reduced to 0 and strong interactions (e.g., scores ... Zuckerberg Initiative. We hereby announce the general availability of new a tool for CRISPR scientists...project is funded by CZI and the CZ Biohub for the acceleration of science. It's open-source. You can see all...analyses, but it also allows you to download any generated info for your own purposes, or delete the originals...that it can be used in the rank equation above. Generally, the greater this distance between the cut and...sequence inserted by HDR. Although this practice is generally advised, there is little detail published on how...

...used for many years for in vivo delivery and gene therapy and represent one of the most studied viruses. ...with a packaging capacity of about 6 kb, the 1st generation of AdVs have already a packaging capacity larger...presumably reduce the likelihood that donor DNA will interact with sporadic double-stranded chromosomal DNA ...and very efficient introduction of DNA into therapeutically relevant, non-transformed mammalian cells. ...developed and used AdV vectors to edit genes in several biological systems. It has been used for instance...deficiency (most widely used so far) or for cancer gene therapy such as nonalcoholic steatohepatitis cancer. In...Targeting. In: Applications of viruses for cancer therapy. Elsevier, pp 39–67 Ding Q, Strong A, Patel KM,...

... Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy. Curr Gene Ther, 13(6), 421–... anecdote goes toward saying that host-virus interactions are an important part of the biology of adenoviruses... the commercial market). Although host-virus interactions likely seem important for any virus, we will...initiating the whole process – such as the viral DNA polymerase – it makes sense that vectorization strategies...oncolytic virus in addition to standard uses in gene therapy and in basic research, like other viral vectors...PMID: 31749409. Harrach, B. (2014). Adenoviruses: General Features. In Reference Module in Biomedical Sciences...novo viral gene expression: Implications for gene therapy. Proc Natl Acad Sci U S A, 95(19), 11377. https...

...sequencing (HTS) data we generate and maintain. It became clear that while several people across teams had...enjoy is discussion of current new and exciting literature. As I transitioned out of academia and away from...researchers of all varieties must stay current with literature and emerging technologies to remain competitive...Microbial Pathogenesis that occurred once a week for several months at a time during the spring and fall semesters...club to begin! Figure 1: Discussing next-generation sequencing at the Addgene bioinformatics journal...teams or to stay abreast of current trends in the literature, sampling a breadth of work from a field (perhaps...wandering monologue?  Assuming the feedback is generally positive and the meeting seemed to be of use, ...

...droplet quality. Using a droplet generator, the sample and the droplet generation oil are moved through small...within the expected range. We have also found that generating the standard curve for qPCR can be challenging...Pipette slowly If you are using a manual droplet generator, you will have to be careful about transferring...transferring your droplets from the droplet generator to the PCR plate. Maintaining a high droplet count is important...sample. Even if you are using an automated droplet generator, you should avoid pipetting too quickly when making...rate of 2C/s is recommended to ensure an even temperature change among all of the droplets, but you can...recombinant adeno-associated viral vectors." Human Gene Therapy ja (2019). PubMed PMID: 31140327. PubMed Central...

...disease. This work will hopefully accelerate the development of targeted therapy for peritoneal mesothelioma...learn how plasmids from Addgene will be used to accelerate rare disease research and provide hope to patients...which disruption of the smn1 gene results in the degeneration of spinal cord motor neurons. This disease is...using C. elegans as a model to identify SMN1 interacting partners and perform whole animal screens for...for small molecules that block the neurodegenerative effects of smn1 disruption. As part of his work in...will use Addgene plasmids to characterize smn1 interactions with other genes. He will use fluorescent proteins...of the molecular biology behind SMN1 and its interaction partners, Dr. Di Schiavi will be able to better...

...markers all tagged with mEmerald. Plasmids to monitor protein-protein interactions through multicolor labeling...scale: Look at the mEmerald variants first, followed by the color variants. The mEmerald plasmids offer a...biology, seeing what interacts with what, what affects what. It's like fixing a Maserati. If we know how all...Fusion Proteins - Making the Right Connection.  mEmerald plasmids and many more! Baird has a helpful tip...the Davidson lab always tagged new proteins with mEmerald first, Baird says. From F-Actin to mitochondria... memerald...

...(2023) Nanobodies: A Review of Generation, Diagnostics and Therapeutics. Int J Mol Sci 24:5994. https:...The UN General Assembly has declared 2024 the International Year of Camelids. The declaration is intended...into the clinic as well. The first sdAb based therapeutic was approved in 2018, with many others in clinical...clinical trials (Jin, et al. 2023). In addition to therapeutics, there is a lot of excitement around using sdAbs... synthetic single-domain antibody libraries to generate new sdAbs. There has also been some interesting... (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable domains of heavy...their impact grow as new sdAbs become easier to generate and more widely adopted. So thank you to the camelids...

... due to the interaction with quizzes and discussion on that week’s topic by the moderators. The assignments...scientists. Over the course of the class, the moderator discussed individual sections of a paper, the ...laborious, but helped build my understanding of how to generate a manuscript. At the end of the online course,...understanding of how to apply it. The personal interaction of critiquing others’ manuscripts as well as ...the writer works.  4. Practice writing everyday Overall, the most important instruction the classes taught.... Over time that portion of the paper will be generated. After you complete a publication quality figure...figure. Writing a scientific manuscript is like generating a quilt with your lab mates, you add the individual...

...and the moderators were on top of any comments that were at all trollish. Kudos to the moderators for keeping...lists of forums on particular topics. Volunteer moderators set the rules for these forums and try to keep...questions! 2. The r/Science subreddit is well moderated. One of our original worries about sharing science...trolls (urban dictionary for trolling: The art of deliberately, cleverly, and secretly pissing people off, ...dialogue). Luckily r/Science has a great team of moderators who facilitated scheduling, filtered out inflammatory... sales-y in posts which falls in line with our general mission to share- as opposed to sell- science. ...expect. Fortunately, Reddit Science is heavily moderated. I started watching the questions and comments...

... Choosing a Cas9 for your knockout experiment Generating a no-fuss genetic knockout  Knock-out cells or...loads of sequences to select from - in many cases several exons’ worth of DNA. Any ∼20 nucleotides of DNA...targeting opposite strands must be available in moderate proximity to each other.  If having two sgRNAs...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...strand break.   Prime editors are the second generation of base modifiers available. This system utilizes...transcriptional activation domain - VP64.  Later generations of activators include the SunTag system which... The Synergistic Activation Mediator complex, generally the strongest activator, consists of a dCas9-VP64...

...and discovery The first generation of CRISPR could cut DNA, the second generation could do base editing,...specificity: when purified, it could be programmed to generate specific cuts on the RNA target. In addition, ...most common RNA targeting system, Cas13, the "collateral" activity of the enzyme can lead to off targets...RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using Cas7-11   Use ...editing, and with the third generation came Prime editing — the ability to now make any small changes, including...small deletions and small insertions, leaving a several kilobase-sized gap in the gene editing field. The...precision into a genome, can be considered the fourth generation of CRISPR, one which drops itself neatly into...

...practiced or required by many other journals and several funding agencies (e.g. Wellcome Trust, NIH Data...information (or links to information) for assessment of veracity of the conclusions drawn. The main difference ...publication. Who is to say that the initial data-generating lab has efficiently trawled their own data? A...much time they had. It could be argued that data generators may, in light of the policy, hold onto data longer... available to everyone at once, including the generators. This practice doesn’t appear to harm the originating... as in an actual publication and not require interaction with the data authors. In some types of research...multiple animals on different occasions via video, generating huge amounts of data. When published, this data...

...Addgene blog Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting... for sharing on the site. The entry form has 5 general question types and it takes approximately 15 minutes...experiment, read our blog post on important considerations when using AAVs. Experimental results: These...virus? If you were using a genetic tool, did it operate as expected? If your experiment did not work, we...Frontiers in Neuroanatomy and summarized some considerations for precise neuronal targeting using AAV on...submission of an experimental image result that you generated with a caption. For microscopy images, please ...microscope used, and objective used. If you did not generate the image yourself and it is under copyright, ...

...kits with greatly reduced costs per plasmid. We generally create kits to allow you to easily use a set of... by making it easy for a researcher to quickly generate N- and C-terminal epitope tag fusions to a protein...protein of interest. The plasmids used to generate these fusions all use the same restriction enzymes for...provided a solution for this problem by developing a general translation initiation sequence known as the Species-Independent...locus-specific libraries compatible with Illumina next generation sequencing in order to study evolutionary dynamics...These bridging primers are amplified and used to generate Illumina sequencing libraries using a 2-step PCR-based...open source platform and the libraries can be generated in a cost-effective manner with minimal bias. ...

...high efficiency CRISPR-based approaches for the generation of knockins via homologous recombination with...approach is most well suited, since it routinely generates knockin efficiencies of 1-40% with G418-selected... CRISPR-based approach. Second, the AAV preps generally used for gene editing are low titre, and are therefore... most current CRISPR-based approaches for the generation of knockins, it is not always ideal. Because ...advantages of the AAV and CRISPR-based approaches, several groups have recently worked to enhance the efficiency...AAV and CRISPR-based approaches is described in several recent reviews (Howes and Schofield, 2015; Moser...PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating human somatic cell gene knockouts using recombinant...