...scientific support staff receives – in fact, the blog originally started as a way to easily store and share in-depth... everything goes through an editing process and final check before publication.   Do research! Nearly...that’s not enough, we consult with experts, watch webinars, or use other research methods. In the case of...to make sure absolutely everything was correct. Final touches We try to use figures and images to help...communicator "just sketch a western blot out quickly." Final design (right). Immediate and immense regret of ...image sharing repositories. Then we format, do a final review to catch those pesky typos, and hit publish...

... Bryan Roth’s lab from the University of North Carolina, Connie Cepko’s lab from Harvard Medical School...provided valuable insight into AAV production. Finally, we are blessed to have an incredibly supportive...” Under the leadership of Addgene’s co-founder, Melina Fan, we created a new team to push this project...project involved every Addgenie in the company. Our Finance, Business and Legal teams had to pave the way for...export and shipping on dry ice. The Business and Finance Teams continue to facilitate the formation of partnerships...dedication to do this well ... because it did. So finally, a big thank you to the Addgenies who went the ...

...others you’ll encounter include monoclonal and recombinant antibodies). By the end, you’ll have a better...column and from the target protein. Pre-adsorption Finally, polyclonal antibodies may be pre-adsorbed to remove...batch variability. Once an animal has undergone a terminal bleed to acquire all of its serum, there is no...polyclonal antibody, or select a monoclonal or recombinant antibody instead. Otherwise, you might experience...of background noise compared to monoclonal or recombinant antibodies. This is because the polyclonal antibody...antibodies Learn about the advantages of using recombinant plasmid-based antibodies Resources on Addgene.org...

...generated with 4 different, binary 2in1 vectors with all possible combinations of tag orientation (NN, NC...activity.   With the addition of blue light, the terminal helices of the LOV2 domain unfold, disrupting ...responds to blue light quickly. Within minutes of inactivating the CASANOVA complex, fluorescent CRISPR complexes...the brain compute using electrical impulses to coordinate emotions, thoughts, and behavior. Historically..., where insertion of the GOI occurs through recombination. The 2in1 cloning system is well suited for...

...depending on the combination of fluorescent proteins expressed. CreLite-mediated recombination was found in...editing efficiencies in the mouse brain, liver, retina, heart, and skeletal muscle. The dosing required...cyanobacteria. This kit contains 96 parts including terminators and promoters, CRISPRi parts, selectable markers...longer present, the fusion proteins separate, inactivating Cre. This is critical for reducing Cre toxicity...various fluorescent proteins controlled by Cre recombination. Each cell can have multiple copies of the transgene...

...websites, Facebook and Gmail, were launched. And Melina Fan, a cell biology graduate student, was frustrated... make sharing reagents and protocols so easy!” — Nina Jain, Harvard Medical School. We want to give a ... really like this award because you don’t get nominated for it, it just reflects how useful the reagents... we could use the same principles to provide recombinant antibodies, another plasmid-based product that... In March 2022, we launched our ready-to-use recombinant antibodies service, part of the Neuroscience ... A blast from the past: Cofounder and CSO Melina Fan setting up Addgene's very first in-house NGS...

...two non-functional fragments: N-terminal BirA* (N-BirA*) and C-terminal BiraA* (C-BirA*). These two fragments...(PPIs). These interactions are often modeled as binary relationships, i.e. protein A interacts with protein...Split-BioID is that it allows for the validation of a binary PPI as well as the identification of additional...complementation assay that can be used to study binary protein interactions. However, FRET generally only...proteins were tagged with. It’s best to test all combinations and pick the conditions that give the strongest...

...general availability of their recombinant antibodies. Since recombinant antibodies are a newer technology...chose monoclonal antibodies (mAbs) and their recombinant forms (rAbs). mAbs and rAbs have higher homogeneity...produce the same results even to a different sample. Finally, as a lab we discuss the results together, explaining...Zhuhao Wu at the Icahn School of Medicine at Mount Sinai.  References and resources References Baker M. ...ValidationSelecting the Right AntibodyPlasmid-based Recombinant Antibodies    ...

...-specific diagnostics for the Ebola virus. Our original proof-of-concept work was exciting because it ...the spread of the virus by the Aedes mosquito (Messina et al, 2016). We quickly assembled a team of students...Asian-, African- and American-lineages). Strain discrimination is especially important in the current outbreak...setting. Molecular diagnostics for the Zika virus predominantly look at viral load in patient blood and urine...self-assembly for applications in health, energy, and sustainability.   References 1. Pardee, Keith, et al. "Rapid...25417167. PubMed Central PMCID: PMC4243060. 4. Messina, Jane P., et al. "Mapping global environmental ...

...Validated recombinant antibody toolbox for aminoacyl tRNA synthetases By Kate Harten Recombinant antibodies...library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases Nanocompartments... axonal projections and putative pre-synaptic terminals with one vector. AAV hSyn FLEx mGFP-2A-Synaptophysin-mRuby...Synaptophysin-tagged GCaMP6s is enriched in the pre-synaptic terminal providing better signal to noise for imaging calcium...dynamics in fine structures. We have expanded our recombinase-dependent virus selection to now include Flp-...

...°C instead of 37 °C and then screen for ITR recombination with a SmaI or XmaI restriction enzyme digest...RecA, a protein that aids in the homologous recombination of duplicated regions like the ITRs. Restriction... concentrations of iodixanol to separate out contaminants from an impure AAV preparation. 15%, 25%, 40%...standard and AAV transfer plasmids. It’s easy to contaminate a workspace with plasmid, not that I would know...adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human gene therapy methods, ...R.A., & Samulski, R.J. (2007). Production of recombinant adeno-associated viral vectors for in vitro and...

...training, going from intermediate to advanced and finally, to expert.   Figure 1. Visual representation...subcutaneous injections take some practice to get the coordination down. Subcutaneous injections are most often...delivered to the central nervous system via cerebral spinal fluid. The injection site is the space in and around...minimally invasive. Restraint: anesthesia Location: spinal cord Gauge: 30 Volume: up to 0.01 ml (10 µl) Skill...what method you’d like to use, remember that the final decision will likely come down to experimental necessity... 26 < 0.05 mL Advanced Intrathecal Anesthesia Spinal cord 30 up to 0.01 mL Expert Intratracheal Anesthesia...

...cells and eliminate them from the body. While effective, the immune system does not always eliminate every...with the goal of being able to discriminately and effectively eliminate cancerous cells from the body..... What is cancer, really? While cancer is extraordinarily complex at the molecular level, at the cellular...commit itself to the apoptotic pathway and be eliminated from the body. In the case of cancer, however...

...stability and yield. If a plasmid is prone to recombination Use a strain optimized for plasmids with repeat...elements, such as viral plasmids with inverted terminal repeats. We use NEB Stable to transform and prep...Stbl3 have been engineered to reduce plasmid recombination, but are also endA+. endA encodes a thermostable...volume of culture used for inoculation if we get inadequate plasmid DNA yields. Why would volume make a difference...recommended range of the filter or you might inadvertently lower the amount of DNA you purify! Additionally...should be determined for each plasmid/strain combination individually, based on determining the cell density...

...developments.  Further, this post may contain technical inaccuracies or typographical errors.  No action should be...patents and letters of recommendation. EB-1A extraordinary ability category The EB-1A - is similar to national...journal editor) of the work of those in your field. Original contributions to the field that have had a major...include the fact that you are a classically trained ballerina who has performed at Lincoln Center. Your case...of permanent, tenured or indefinite (no fixed termination date) employment at a university or institution... the same or allied academic field Evidence of original scientific or scholarly research contributions...management, alliance management, business development, finance, and operations. To learn more, visit www.propelcareers.com...

...was over 300 years ago. Teaching by example No examination of the state of science would be complete without...story has its own Franklin in Doug Prasher, who originally isolated the GFP gene but was not included amongst...spends some time on Addgene’s role in the rapid dissemination of these technologies, proposing that “Addgene...undermining of the scientific process and the marginalization of science, particularly public health and ... observations and suggestions are not so much inaccurate as already somewhat dated. Zimmer could not have...continue to be a “critical consequence of ...the marginalization of science,” we know now in the summer of 2020...

...alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek et al., 2012... mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting...this form, the CRISPR complex, consisting of a recombinant dCas9 protein and synthetic gRNA, is incubated...DUX4, a transcriptional regulator that when inappropriately expressed can cause facioscapulohumeral muscular...non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing." PLoS One 10.4 ...chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4...

...a wash solution removes contaminants while nucleic acids stick around. Finally, purified total nucleic...sample can be treated with RNase to remove RNA contamination and you can use a phenol chloroform extraction...extraction to remove remaining proteins and RNase. Finally, DNA is precipitated with alcohol and resuspended...into this mixture to absorb the nucleic acids. Containinants bind cellulose less tightly than nucleic acids...with 1M phosphoric acid and re-used, but trace contamination could interfere with interpreting the results...

... digital PCR is a sensitive assay, so cross contamination into a no template control (NTC) well is common...reduce aerosols that could potentially lead to contamination. Optimize your PCR For a good starting place...place on what PCR parameters to use, see Lock’s seminal paper on AAV titration (Lock et al., 2014). If you...tool for titration and integrity evaluation of recombinant adeno-associated viral vectors." Human Gene Therapy...PMID: 29548643. Lock, Martin, et al. "Absolute determination of single-stranded and self-complementary adeno-associated... PubMed Central PMCID: PMC5915492. Witte, Anna Kristina, et al. "A systematic investigation of parameters...

...gene-transfer tools in the study of the brain and spinal cord, which together are known as the central nervous...transduction of both the CNS and PNS.That is until the Gradinaru Lab at Caltech stepped up to the challenge. Find...is the CREATE method?  CREATE stands for Cre Recombinase-based AAV Targeted Evolution. In this technique...short DNA sequences that contain all possible combinations into a region of the cap gene. In this special... one RGB AAV and red, green, or blue are the predominate colors expressed (figure 4c).   AAV Vector...therapy treatments for neurological diseases). The Gradinaru Lab has a lot going on. Here are the key takeaways...