We narrowed to 877 results for: HA

...targets by: Mike Lacy Adding to their set of anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and...iRFP713, SNAP-tag, and HaloTag fusions, while the anti-HA frankenbodies include mEGFP, mCherry, and HaloTag...Adapted from Liu et al. 2021. Anti-FLAG and anti-HA frankenbodies have a variety of applications, including...blotting, and more. Given the popularity of FLAG and HA tags, you may not even need to clone new constructs...requires a small epitope tag (FLAG is eight amino acids, HA is nine), multiple repeats of the tag can recruit...larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic screens because...

... expressing IgM. IgM has a pentamer structure, which means each IgM molecule has a binding capacity or...function is unknown. It has a monomer structure with a valency of two and has no subclasses. IgG IgG is... appropriate isotype control. An isotype control has the same constant heavy chain domain as your primary...replaces the hinge region in IgM antibodies. IgM has no subclasses and is found mostly in blood. IgD While...

...communities and welcome suggestions in the comments.  X has moved significantly away from the functionality and...conversation in the scientific community. Addgene has been considering a gradual move off X as a response...conflict with our values. For this reason, our timeline has accelerated. We significantly reduced posting on ...

...Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while GeoCas9 has maximum activity from 50...the recent discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR...stability in human plasma The last trick GeoCas9 has up its sleeve is its increased longevity in human...stable when incubated in reaction buffer alone, but has limited activity when incubated in diluted human ...

...You will also have to make sure that your plasmid has been incorporated into the host organism, usually..., c-Flag pcDNA3, comes from the pcDNA series. It has the CMV promoter for mammalian expression, a multiple...mammalian selection cassette (e.g. 1435 pSG5L Flag HA). You can also upgrade your basic mammalian expression...considered to be a more robust expression system and has its advantages; however, remember that you will need...the specific mutations. For these purposes Addgene has a large collection of yeast expression vectors with...

...sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting ...genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose. Epigenetics began...many TALEN-based technologies, the advent of CRISPR has made targeting much easier! With the help of guide...modification at specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs including...BioRender.com. Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-CD for targeted cytosine demethylation...Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš’ lab has deposited pdCas9-DNMT3A-EGFP and pdCas9-DNMT3A-PuroR...DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion...

...until 2005 [1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The...offers a large cargo-carrying capacity (over 200 kb has been demonstrated) with no known upper limit [1]....particularly useful when the gene being expressed has threshold effects, such as with signaling molecules...transgenic cell line with the piggyBac™ system is that it has a preference for integration at regions of open chromatin...removal, which alters the endogenous DNA sequence and has been suggested to cause cytotoxicity [5]. Second,...efficiency. While the efficiency of excision with PBx has been shown to be higher than that of the wildtype...Cre recombinase, the PBx is highly specific, and has not been shown to cause genomic abnormalities when...

...mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector that can be ...and the subsequent biochemical characterization, has enabled scientists to engineer a wide variety of ...green to red when it is oxidized. The Timer tool has already been used by several groups to monitor protein...imported to the mitochondrial matrix. Gottlieb’s lab has used an inducible MitoTimer reporter using a Tet-... for protein-peptide locking, Mark Howarth’s lab has developed a new tool for peptide-peptide locking....optimization through engineering, Ed Boyden’s lab has identified a variety of new channelrhodopsins for...nicknamed Chronos, is blue and green light-excitable and has faster kinetics than those of other channelrhodopsins...

...between a cell that has had one viral particle integrated into its genome and a cell that has had multiple integration...The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected... be significantly higher than the same prep that has been stored in the freezer. If you plan to freeze..., you may want to consider using an aliquot that has been subjected to a freeze/thaw cycle for titering...CRISPR Genome-Wide Screening Learn How Lentivirus Has Been Used for Lineage Tracing Additional Resources...

...science has become increasingly restricted to academic and industrial labs, but recently there has been ...KittyBiome has been incorporated as a non-profit entity and so far most of their funding has come from ... the predictions about the effects this mutation has on locomotion in our computer model to the effects...Genetics Center (CGC) at the University of Minnesota has declared our makerspace an educational institution...

...to define how you will know that the relationship has been successful – what are the desired outcomes? ... Dora Farkas, founder of Finish Your Thesis (who has also blogged for us) offers a free report on her ...called “Secrets to Success in Graduate School”. She has also launched an online career coaching program that...in Madison, Wisconsin she also works remotely and has experience as a scientific recruiter. Chris Humphrey...of From PhD to Life. She runs great tweet chats, has an outstanding blog and, while her PhD is in History...remotely, will travel for speaking engagements and has many areas of specialty from leadership development...

...have the biggest impact. We should know — Addgene has built its foundation on this idea! And we believe... cell populations and regions of the brain. This has culminated in a collection of AAV enhancer plasmids... to express exogenous genes — a feature the AIBS has capitalized on. Figure 1: Schematic of transcriptional...Allen Institute’s Genetic Tools Atlas (GTA). The GTA has a wealth of information and includes annotated images...target a very specific cell type. However, Addgene has partnered with the AIBS to showcase the “best-in-...More from the Allen Institute The Allen Institute has many other projects outside of the enhancers and ...research is conceived, developed, funded, and shared has no doubt changed drastically over the past few decades...

...in living cells. The HA frankenbody is an HA tag detection probe. It contains an HA-binding scFv that’s...often genetically fused to other proteins, which has led to their application for basic and translational...fluorescence intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged...s fused to a fluorescent protein such as GFP. The HA frankenbody works just like an antibody-based probe...of cytotoxic granules from the T cell. When a CAR has an scFv that binds cancer-related antigens, binding... Addgene blog: Read about frankenbodies, an anti-HA scFv-based probe for fluorescent imaging. Learn more...

...also the fastest test that has been developed so far.  A similar method has been optimized to detect the...Two-step-SHERLOCK has a sensitivity in the zeptomolar range while One-step-SHERLOCK has a sensitivity in...State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...full-fledged pandemic. Containing the spread of this virus has become an utmost priority for countries around the...the outbreak ultimately flattening the curve, it has become evident that widespread testing is crucial... 83 known COVID-positive samples tested, DETECTR has a positive prediction rate of 95% and a 100% negative...

... dark but dissociate under blue light. This tool has now been modified by Torsten Wittmann’s lab at UCSF...genetic or pharmacological manipulations on cells, and has potential use for a wide range of applications in...monomeric, 4-5 fold brighter than mNeptune684, and has a favorable pKa of 5.6. To test out this new FP in...The increasing popularity of single base-editors has resulted in the rapid expansion of the genome editing...expansion of genome editing tools, the Reuben Harris lab has deposited a reporter plasmid that allows for rapid...

...There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing field ...CRISPR Therapies Cancer and CAR-T therapy The FDA has already approved CAR-T immunotherapy for cancer treatment...CRISPR Therapeutics. Data shared from these trials has been outstanding, with almost all patients experiencing...desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One treatment...way. Delivery, especially for systemic approaches, has its own unique set of obstacles. Thus far, several...delivery tends to be trafficked to the liver. Thus, it has been used for hATTR therapy, since TTR is primarily...

... It was inspiring to hear how the genome project has already had a major impact on undiagnosed and rare...its work on the origin of cellular life. His lab has long hypothesized that life started with RNA and ...prokaryotic genome. Then he desribed how P. furiosus has systems to defend against both DNA and RNA viruses...technical developments. Using the Tet-on system, his lab has created a tetracycline cas9 inducible knock-out system...in a complicated conformation “not like DNA which has a boring structure.” That certainly summed up this...this conference—nucleic acid science still has many surprises for the researchers in this field. Resources...

...enormously plastic tool and has taken the scientific world by storm. While Cas9 has been most widely used to... to create specific edits in DNA, there has also been significant work on constructing Cas9 transcriptional...CRISPR activation for genes with known functions has the potential to be used in therapeutics, as it can...changes in phenotypes. For example, CRISPR activation has been used to differentiate induced pluripotent stem...creation of innovative technologies. To date his group has develop several popular CRISPR-based tools for altering...Genome-wide Screens with CRISPR/Cas9 Learn How CRISPR Has Been Used with AAV Resources on Addgene.org Find...

...Recently, Xue Sherry Gao’s lab at Rice University has developed DAP arrays for efficient multiplex base...process and release its own gRNAs from a single array, has been a top choice for multiplex gene knockout or ...proper functions to enable efficient base editing and has yet to be developed for prime editing, both and their..., which is conserved in all living organisms and has previously been used for multiplex Cas9-nuclease ...lengthy promoters, such as U6 or EF1α promoters, has limited the number of gRNAs that can be packaged ...

...to realize that the effect of Dr. Yamanaka's work has been profound, and this is a rare instance in which...address, appeared almost incredulous that the field has exploded so quickly, citing the first clinical trial...scratch the surface of different fields. This meeting has given me an opportunity to dive a little deeper into...scientists care about patients As any grad student who has spent years studying how that one protein interacts...presenters, it was gratifying to see that Addgene has ensured that plasmid acquisition is not a rate-limiting...

...biology. The mistrust and anger directed at GM crops has burdened this emerging engineering technology. Monoculture... metabolism are well understood. Additional work has also identified and characterized mechanisms and ...petunias. Because of the extensive research that has come before us, it’s possible for us to combine these...plant synthetic biology. Plant molecular biology has traditionally relied on overexpression and knockouts...Nikolai Braun grew up outside of Washington D.C. and has always enjoyed exploring the outdoors and experiencing...

...technology has revolutionized the fields of molecular biology and bioengineering, as it has facilitated...site, or protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one .... When native Cas9 is complexed with a gRNA that has been truncated such that it exhibits 15 or fewer ...truncated gRNAs Importantly, the use of truncated guides has largely been demonstrated to result in decreased ...creation of innovative technologies. To date his group has develop several popular CRISPR-based tools for altering...

...considering my future path. Talking to Addgenies has helped me get a much better idea of what lies ahead...Director of Product Management Eric Perkins, who has been in many roles in Addgene since he started, said... set up for failure. Listening to Kenkel’s story has helped me realize that is not true at all. Racheal...medical school was devastating. At the same time, she has an entirely new path to discover and gives her time...finance, and while I've done that, my big takeaway has been the impact I can have on an organization beyond...was one of the biggest takeaways from my co-op. It has been so insightful to learn about different career...

...positions. There has been a lot of engagement with the Athena SWAN Charter and it has really strengthened...their pathogens. As Patron explains it, her work has always been focused on gene transfer, from transgenes...MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate and enable plant science...quantitative data for regulatory elements, which has not been available for plants. Addgene: Will the ...strengthened the network of female scientists. Twitter has been really good for that too. I’m more in touch with...

...proteins such as Green Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish for...from the crystal jellyfish Aequorea victoria. It has a fluorescent emission wavelength in the green portion...protein isolated from Discosoma sp. Similar work has been done to expand the red-fluorescent protein repertoire.... Split EGFP: An alternative to FRET, split EGFP has also been used to study protein-protein interactions...A wide array of GFP-based fluorescent biosensors has been designed to detect a variety of intracellular...surface of the potential applications for GFP. It has also been used to identify particular cell populations...

...It’s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and ...human genes. Unlike the GeCKO library, each library has been enriched for different gene targets. These pooled...system. Genome wide mouse CRISPR library The Yusa lab has leveraged the CRISPR system to target all 19,150 ...stably transfected for Cas9 expression. The Yusa lab has used their library to screen for genes involved in...

...five years, inflation has occurred. Here at Addgene, the consumables cost per stab has increased 50% since...make a plasmid. Doench’s lab in the Broad Institute has created hundreds of thousands of plasmids, using ...since 2019, while overall inflation of USD has gone up 19.8% in the same time period. For ease of calculations...per plasmid, Addgene, since its founding in 2004, has saved the scientific community a collective and very...

...sharing open hardware has a similar goal. The sharing of modules, ideas, and blueprints has the potential not...Since its early years, the light microscope has been a powerful tool in almost all disciplines. In biology...Sharing open-source plans that rely on such tools has allowed the community to regularly replicate designs...portable device for studying plankton using flow cells, has become an established alternative to high-priced ...

... funding model in nuclear receptor research, and has generated a set of comprehensive datasets that collectively...interactome of these key regulatory molecules, and has provided clues to their previously uncharacterized...website. NURSA and Addgene The great progress that has been made in the NR field over the last thirty years...and coregulators, and although the reporter gene has over the years evolved from chloramphenicol acetyltransferase...research tools is a double-edged sword, however, and has greatly increased the burden associated with identifying...

... the best outcomes. The go-to in vivo option AAV has become the preferred delivery method for in vivo ... other viral vector delivery methods. Lentivirus has limited packaging options, with most lentiviral vectors...possibilities when designing screens. Each serotype has its own tropism, or a preference to infect specific...BioRender.com. Beyond the identified serotypes, AAV also has psuedotypes. Psuedotyping is the process of making...Lentiviruses have capacities of 8–12 kb, while AAV only has approximately 4.7 kb — which means that when using...the genetic landscape of tissue-specific diseases has grown exponentially, aided by advancements in in ...

...repository. Our blog is just over a year old, and it has grown steadily during 2014. We were excited to have...continues to grow as the genome editing technique has developed and improved. The CRISPR posts written ...about. Our post on “Making Your Own Competent Cells” has been extremely popular, generating many additional...

... applications (v82 has higher affinity for glutamate but slower kinetics; v857 has faster kinetics but...iGluSnFR3 (Aggarwal et al. 2022). This new version has higher signal to noise ratio and is better localized...for two-photon microscopy. The resulting JEDI-2P has faster response kinetics and increased brightness...and deep-tissue imaging. Vladislav Verkhusha’s lab has generated a new genetically encoded calcium indicator...interest, it’s important to choose a biosensor that has a binding affinity close to the range of concentrations...physiology and metabolism under various conditions. LiLac has improved response size and reduced sensitivity to...

... but inadequate access to reagents and equipment has become a bottleneck. In the last few years, scientists...nucleic acids at the atomolar level. The Zhang lab has adapted natural RNase activity of the Cas13 protein...Gootenberg et al., 2017 and 2018). While the Doudna lab has used Cas12a’s non-specific ssDNA degradation to develop...field (Myhrvold et al., 2018). Now, the Zhang lab has shared a protocol for using SHERLOCK to detect SARS-CoV...at atomolar levels, within one hour. DETECTR thus has the ability to rapidly detect nucleic acids with ...diagnostic applications. The company Mammoth Biosciences has been started based on DETECTR technology with the...Biotechnology. Like the SHERLOCK detection system, DETECTR has not been approved for diagnostics yet, but is undergoing...

...specific protein folding needs. The pCri system has recently been used to study a matrix metalloproteinase... Queensland’s Institute for Molecular Bioscience has provided a solution for this problem by developing...eukaryotic cell extracts (Gagoski et al 2015). He has also constructed a library of eGFP-tagged human ORF...constructs. The Cell-Free Protein Expression Kit has been used to produced recombinant proteins for in-vitro...populations through time, Christopher Marx's lab has engineered and validated a new method called FREQ-Seq...cost-effective manner with minimal bias. The FREQ Seq kit has been used in microbiology studies monitoring sequence...binds your RNA sequence of interest. The Zhao lab has made this easy with their PUF Assembly Kit. The kit...

...graduate program at the University of Manitoba. Our lab has been utilizing chemogenetics (Designer Receptors ...Although AAV2 can transduce many tissue types, it has low expression in several brain regions where other...serotypes to infect different cell types. In the CNS, it has been shown that there is variable transduction of...from the Original Location - Axonal Transport It has been demonstrated that certain serotypes can undergo... of movement, distance traveled, etc.) (4). This has also been identified with AAV2 (5), AAV6 (6) and ...required for the onset of gene expression. However, it has yet to be determined what gene expression looks like...problem in many experiments, a group of researchers has identified this feature of AAVs as a new tool for...

...excision site after cleavage by the transposase. It has a cargo capacity >100 kB, although integration efficiency...efficiency decreases with cargo size. Sleeping Beauty has a close-to-random integration profile in mammalian...hypersensitivity sites. Like Sleeping Beauty, piggyBac has an integration efficiency in human cells similar ...1996). Unlike Sleeping Beauty and piggyBac, Tol2 has a weak consensus sequence for its preferred target...hypersensitivity sites. Tol2 is only active in vertebrates and has lower efficiency of integration in human cells than... is a truncated version of the original Tol2 and has ~3-fold increase in transposition activity  Tol2...encodes a Type I-F CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA (crRNA), 2...

...Research will be held from 22-24 October. This blog has been contributed by guest blogger and Future of Research... of a system that, despite all of its successes, has many deep-rooted flaws (1). As we discovered through...example, Harvard’s Therapeutics Graduate Program has a compulsory internship to be taken in an industry...Marder, professor of Biology at Brandeis University, has recently published an article arguing against reducing...Labor and Worklife Program at Harvard Law School. He has continually promoted the cause of increasing data...

...methods. Each method has its own pluses and minuses, but Golden Gate cloning has been especially useful...number of potential clones. Golden Gate assembly has a few advantages over other cloning methods. Exonuclease-based...just a few steps. More recently, CRISPR technology has adapted Golden Gate cloning for inserting the appropriate...also be adapted to express multiple gRNAs. Addgene has two Golden Gate-based gRNA assembly methods available...

...THE ORIGINAL NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED ...Natronobacterium gregoryi, or NgAgo for short. Addgene has already distributed this plasmid all over the world...Publication at Addgene Cas9 and NgAgo head to head NgAgo has a number of unique features compared to Cas9. These...compare CRISPR and NgAgo, but it seems that NgAgo has lower potential for off-target effects. In Gao et...

...2015 has been an exciting year for the Addgene blog. We’ve released over 80 posts on a diverse array ...Scientists and the Plasmids 101 2nd ed, the 3rd edition has now been released), and have started providing recorded...incredibly happy to see that one of our career posts has made it to the top of the list this year. While we...doing just that. Do you like the direction the blog has taken over the past year? Do you think there are ...