... and decide whether you will create an N- or C-terminal fusion based on your experimental needs. Equally...have reported that FP cysteine residues form inappropriate disulfide bonds in the secretory pathway (Figure...localization. For example, the formation of inappropriate inter-chain disulfide bonds between FPs blocks...and blue variants that can be used in various combinations. For example, with standard fluorescent microscope...can label multiple proteins of interest with a combination of green and blue or cyan and yellow moxFPs. ...postdoctoral fellow at the University of North Carolina at Chapel Hill. Under the co-mentorship Blossom...

...  The Addgenies will choose up to four finalists, and the final winner will be determined by popular vote...cloistered consideration, and with much debate, we have finally narrowed it down to three choices.   Fig...unveil the winning name on Feb. 24. Stay tuned!    Original Blugene and Aavery would like your attention, ...Additionally, anyone whose submission is included in the final four will receive some rockin’ newly designed Addgene...

...-open. In the two closed states, integrins are inactive, with low affinity for ligands. When integrins...toolset IPI’s anti-integrin toolset comprises 26 recombinant antibodies (rAbs) either produced de novo using...into two groups. Seven are reconstructions of the original hybridomas as new reproducible rat or mouse rAbs.... Based on the established applications of the original hybridoma monoclonals, we anticipate that our ...other RGD-binding integrins, individually and in combination, showing that among the three αV integrins expressed...an expanding collection of readily accessible recombinant antibodies and plasmids. To encourage collaboration...

... 37°C heat block, LED blue light illuminator or UV light illuminator. Approach: Highly similar to the ...Cas12a activity gets activated. This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied...extracted from a sample and subject to isothermal recombinase polymerase amplification (RPA) using a commercially...amplification is done at one constant temperature, eliminating the need for thermocyclers. This is followed ...contains SSB, strand displacement DNA polymerase, recombinase, and the ssDNA-FQ reporters that exhibit fluorescence...

...protrusions and overlaps (arrows) after blue light illumination. Scale bar 25 µm. Images adapted from Mahlandt... 10, 30, 50, and 100 times the activity of the original, generated using directed evolution (Tei et al... can hydrolyze phosphatidylcholine (PC), the predominant membrane lipid, to phosphatidic acid (PA) or ...GFP-PASS (green). After 30 minutes of blue light illumination, substantial PA accumulates in the plasma membranes...other enzymes. Conclusion We hope this post has illuminated some of the many tools available for controlling..., & Hehnly, H. (2023). Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right...

...Nobody EVER complains that a seminar was too easy to understand. Make your seminar accessible. Let the experts... at [email protected]." You can find his original blog post below! --Ed.  Eight years ago, I decided...proper consideration. 3. Present an easy to follow seminar See item 2. Attendees will include grad students.... This isn't the time to go into detail. Finally, state how successfully completing the research...applying for It's not about you and how you will finally be granted the faculty position you have craved...

...short (1 kb) homology arms underwent homologous recombination at a frequency three orders of magnitude higher...strand invasion, a key step in the homologous recombination pathway. The Waldman lab at the Georgetown University...for the generation of knockins via homologous recombination with a homologous repair template. It is this...endogenous epitope tags, or the introduction of dominant mutations of a gene into the endogenous allele... mechanisms and consequences of cohesin gene inactivation in human cancer.     References 1. Russell, ...generating human somatic cell gene knockouts using recombinant adeno‐associated viruses." Nucleic acids research...

...1977, Sanger sequencing, also known as chain-terminating or dideoxy sequencing, was a major breakthrough... of all possible lengths because extension is terminated upon the incorporation of a ddNTP. Researchers...gels are classic techniques that allowed for many seminal scientific discoveries (5, 8). While newer techniques...techniques. For example, restriction mapping led to recombinant DNA technologies that are now commonplace in ... Alan R. Coulson. "DNA sequencing with chain-terminating inhibitors." Proceedings of the National Academy...al. "Large-scale and automated DNA sequence determination." Science 254.5028 (1991): 59-67. PubMed PMID...

...transferred to a membrane, stained with antibodies, and finally visualized.   This process allows you to detect...typically do not transfer as well using this method.  Finally, commercially available dry blotting systems efficiently...materials and methods sections of those publications.  Finally, always verify that the chosen antibody works in... these cases, the western should be repeated.  Finally, while westerns are a useful way to gauge protein...phosphorylation, acetylation, methylation and ubiquitination. For example, to test if a protein is phosphorylated...and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112:195–...

...the billions of dollars (Freedman et al 2015). Finally, as Pamela Hines of AAAS points out on the panel...enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments Researchers...so-called “negative” results that conflict with our original hypotheses. Looking for novelty in our stories...similar subjects in a single, searchable space. Finally there are new publishing outlets like F1000, PeerJ... presents only the results that align with the original hypothesis. Other practices that fall into this...researcher would get in-principle acceptance of the final manuscript regardless of whether the ultimate results...

...Gautier et al., 2008). Although based on the same original enzyme, SNAP-tag and CLIP-tag show good specificity...(Erdmann et al., 2019). Check out our recent Illuminating Choices blog post for more discussion about ...Allgeyer, E. S., Erdmann, R. S., Wood Baguley, S., Sirinakis, G., Schepartz, A., Baddeley, D., Toomre, D. K... R. Y. (1998). Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. Science,...Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana...from Addgene Better Dyeing Through Chemistry Illuminating Choices: A Guide to Selecting Fluorescent Dyes...

...you see that there is high RNA expression in the retina and in parts of the brain, but almost none in the..., you will see a strong antibody signal in the retinal sample, but little to no signal in the liver sample...CRISPR/Cas9 to create mutations that essentially eliminate expression of the target. Removing (or reducing...interest in your sample, should lead to reduced or eliminated antibody signal. If you see a similar antibody...non-mutant line but no signal in the mutant line! Recombinant Strategies This approach is essentially the opposite...ready-to-use antibodies from Addgene Designing a recombinant validation experiment? Find plasmids here!  ...

...exchanged contact with Amy C., Addgene’s intern coordinator shortly after hearing her talk about Addgene’...program, teaching me the basics of physics and sustainable design while allowing me to do my internship ...results every week always came with surprises. Originally we thought during the first three weeks that ...capstone project report and presentation, during the final week at Addgene. This report shared my work by breaking...each day, my background, observations, etc. On my final day (today!), I presented my research to Addgenies...

...accessibility of CRISPR-based phenotypic screen datasets. Original published CRISPR screen data is curated and deposited... more than 45 publications, beginning with the seminal research articles that demonstrated the feasibility... Each screen curated in ORCS is linked to the original publication via its PubMed identifier, which can...metadata.   Screen results are reported as the original quantitative gene-level scores provided by authors...rather than sgRNA-level sequence read scores. The original supplemental data tables available with the publication...

... PMID: 30678704. Sparse cell labeling using recombinase competition Article contributed by Aliyah Weinstein...Weinstein Sparse Predictive Activity through Recombinase Competition (SPARC) is a fully genetic tool for...organism. The SPARC system uses competitive recombination sites to selectively remove a stop cassette ...effector is expressed. By truncating one of the recombination sites in the construct to different lengths,...monitoring in vivo. To overcome these technical issues, Sabrina Büttner’s lab designed a calcineurin transcriptional...

... the protein with enterokinase, which recognizes the 5 amino acids on the C terminal of the tag. Of the...background can be a huge challenge. Epitope tags eliminate the need for target-specific antibodies and have...genomic locus or more commonly via cloning of recombinant proteins. Why use a tag? If you are fortunate...protein that can also be used as a tag! GFP was originally derived from jellyfish (Aequorea victoria) and...

...conversion is a unique characteristic of recombinant antibodies. Recombinant antibodies are plasmid-based and ...The resulting chimeric antibody would have the original variable regions from the parental rat antibody...antibody retains the binding specificity to the original target and can be used with secondary antibodies...is routinely used to increase the utility of recombinant antibody tools, the resulting chimera may have...

...single-cell sequencing approaches, the lab was able to eliminate the need for axenic (pure) laboratory cultures...distance. At each collection site, we took GPS coordinates, water temperature readings using a heat gun,...stabilization of unknown bacteria. Figure 2: Christina Mork collecting samples at the hot spring. ...leave your samples behind! Paul and Anastasia (Potanina) Neddersen characterized a few samples by 16S ... Christina Mork...

...by Klaus Hahn's lab at the University of North Carolina (plasmids at Addgene). It consists of two domains...and as a consequence, EB1 protein function is inactivated by the separation of its synergistic domains....and colleagues started with the FP mNeptune684 (originally described by Li ZY, et al. PLoS One, 2016) and... editors that utilize dCas9 fused to cytidine deaminases like APOBEC. To achieve this they created a lentiviral...efficient than the BE3 plasmid containing rat APOBEC1. Finally, they show that the ACE reporter can also quantify...

...In our case, we will add TAAGCA, resulting in a final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC...amplification. We can start similarly, taking the final 18 bases of the ORF, including the stop codon (5...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA...restriction cloning, it is essential that you verify your final plasmid by sanger sequencing. DNA replication by... you use, it is important that you sequence the final product. Additional Resources on the Addgene Blog...

... your protein of interest will only occur in combination with that reporter, you can infer its presence...using a mix of different antibody-fluorophore combinations for different markers of interest, you can separately...for distinguishing live from dead cells. The combination of antibody-fluorophores, fluorescent reporters...BV421 and RB545. Fluorophores can also come in combination of two conjugated single fluorophores, like APC-Cy7...be emitted by either of the two. As a result, inaccuracies in the detection of fluorophores (and the markers...should try to avoid or reduce it whenever possible. Finally, we designed a simple panel to analyze transduced...

...atypicalities, including a patient with amusia - the inability to perceive music as anything but noise. As a ...pop psychology, Musicophilia is accessible and fascinating, all without compromising on the relevant science... Tsang, Science Communications and Marketing Coordinator Non-science favorites  The Mistborn series by...even more sinister. 1000 pages long. Incredibly imaginative, funny, bittersweet, and shocking at times."     ... PhD, majored in Biochemistry or International Finance. A book club could lead to smart, sometimes contentious...

...demonstrated in different cellular environments such as retina or glioblastoma). They also found that exo-AAVs... protects them from antibody recognition and inactivation (György et al., 2014), but scientists have yet...deafness (György et al., 2017), exo-AAV2 in mouse retina after intravitreal injection to transduce bipolar...difficult to reach, and are more resistant to inactivation through anti-AAV antibodies. References György...vector mediates robust gene delivery into the murine retina upon intravitreal injection." Scientific reports...

..., the problem comes down to inadequate information about what the original researchers actually did. Admittedly... have to be this way. We can never completely eliminate the risk that a minor variation in experimental...put down a basic outline and refer back to the original paper that everyone in your lab has been referencing...President of Editorial at Cell Press.  She trained originally in cell biology, completing her PhD at the MRC...

... pathway disrupt target gene sequences (thus inactivating genes), while breaks repaired by homology directed...the biological cause of this repair bias is a fascinating (and yet unanswered) question. Our recent paper...identical dissociation rates for catalytically inactive dCas9 molecules. We followed these experiments...vitro results also demonstrated that catalytically inactive dCas9 produced a “bubble” structure in uncut target...homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA." Nature...

...goals require methods to directly activate and inactivate biological processes. Chemical effectors are ...two-component systems (TCSs). TCSs consist of a histidine kinase that phosphorylates a response regulator (RR). ...from cyanobacteria to a common E. coli histidine kinase. This hybrid construct senses light, and an obligate... two-component system (TCS). A hybrid histidine kinase/photosensor senses various light inputs, and an...issue; some promoter activity persisted in the inactive state. The dynamic range, defined as the difference...

...so it’s better to start over if that happens.  Finally, if you’re seeing a weird issue and can’t figure...ladder compatible with your imaging system; or a combination of the two. There are many commercially available...SDS for your proteins to migrate…so don’t try eliminating one component entirely. If you are trying to ...bit to find a membrane, transfer, and buffer combination that works. Background If you’re seeing odd background...manufacturer. You can also consider using monoclonal or recombinant antibodies, which have much lower lot-to-lot ...point. You can also consider using polyclonal or recombinant multiclonal antibodies to increase overall signal...enough antibody binding sites Polyclonal or recombinant multiclonal antibodies Protein not properly...

... the synthetic biology scene in 1989 through a seminal paper from Peter Schultz’s lab describing a technique...same for all three components, meaning that the final protocol may involve a sensitive balance of trade-offs...GCE4All is a great place to start. They host monthly webinars, workshops, and a bulletin board on their website...their first protocol is available now. It is for recombinant expression of phosphoserine-including proteins...Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli. 12(21). https:/...

...washing and then incubate with Proteinase K to reverse the crosslinking. Finally, purify your DNA with a PCR...fragment size and (2)  incubate with RNAse and Proteinase K to purify the DNA and then determine concentration...isolated your antibody:protein:DNA complexes and finally (5) isolated the DNA from your antibody:protein... on Addgene.org Find plasmids encoding recombinant antibodies at Addgene ...

...their target protein is altered by blue light illumination. This ability to reversibly bind and release...tagged, which limits these approaches to use with recombinant proteins. Purifying an untagged protein with ...initiates the extracellular signaling-regulated kinase (ERK) signaling cascade. This signaling is a lot...signaling by using a fluorescent reporter for Erk kinase translocation. These results demonstrate that OptoNBs...pathway. What’s next for OptoNBs? While OptoNBs illuminate a path to light-controlled protein binding, they...