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...signaling molecules to identify each protein of interest, this gel-based method identifies proteins solely by... blog posts, then you likely already have a good idea of the basics of immunoimaging. A scientist conjugates...place over multiple days. Increased run time is considered one of the major drawbacks of the indirect method...interest - the secondary antibody needs careful consideration before selection. If all the primary antibodies...themselves able to approach immunoimaging assays more confidently, collect a broader array of data, and occasionally...

...recently identified Cas12f systems from uncultivated archaea are much smaller and would be ideal for such...different labs have recently published work that provides a solution to this problem. A miniature CRISPR-Cas...editing with a hypercompact Cas12f1 and engineered guide RNAs Yong-Sam Kim’s lab also created a miniature...Biotechnology paper showcases an improved design of the guide RNA that caps the 3’ end with a structured RNA motif...sheet Resources on Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited...

...degradation. Remarkably, small molecule degraders provide distinct advantages over pharmacological inhibitors...Nabet et al., Nature Chemical Biology additionally provides detailed information on cloning procedures used...molecules by conjugating a CRBN-binding ligand (thalidomide) to an FKBP12F36V binding ligand (ortho-AP1867...commercially available in the near future, and we will provide an update with vendor and catalog numbers. As we...or Jay Bradner ([email protected]). Considerations when getting the dTAG system up and running...while quantitative proteomics experiments will provide the best confirmation of selective FKBP12F36V fusion...

...assembling the toolbox needed for anyone to begin considering genetic engineering projects in a very big way...limitless number of DNA binding proteins with Cas9 guided by different promoters. The challenge in building...therapeutics, if you want to program a cell to identify and target a malignant cell, then you’d need the.... You couldn’t do it without repeating the same guide RNA twice. The second problem was the shape of the...also difficult to build larger circuits with many guide RNAs connected because the signal degrades at each...now? Voigt: We are working on this now, we have ideas, but so far nothing that has worked. I don’t have...

...scientists’ pockets. They allow researchers to identify and isolate very specific biological targets. ...can then be used by thousands of scientists to confidently choose reliable and consistent antibodies for...antibodies, creating a potentially unlimited supply of identical antibodies (See Fig 1.). rAbs are a relatively...collaborates with these companies to create and provide general availability of their recombinant antibodies...creating a database for goldilocks antibodies and, alongside that, a publicly available 2D/3D mouse atlas from...atlas will enable scientists to have an intuitive guide for checking their staining and neuroanatomy without...

...that are around 20 base pairs long. Using a single guide RNA (sgRNA) to create a double-strand break works...non-coding RNAs (lncRNA). lncRNAs are over 200 nucleotides in length and lack an open reading frame, so ... a number of different options. By carefully considering your ncRNA of interest, you should be able to...references Resources on addgene.org Addgene's CRISPR Guide Addgene's CRISPR 101 eBook CRISPR resources More...Doudna JA, Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity...051410-092902 Liu Y, Cao Z, Wang Y, et al. Genome-wide screening for functional long noncoding RNAs in ...

... religious affiliation, and ethnic or personal identity. We do this through a variety of activities, including...experiment, the case of Henrietta Lacks, the Havasupai incident, and the sterilization of women in marginalized...with different organizations and individuals, to identify ways of collaborating, and to organize events ...Having a broad interest and awareness of issues outside of your field of research will allow you to better...better engage these diverse audiences and to identify issues or topics that are matters of concern for ... future work in these areas, and in helping you decide whether you enjoy this kind of work. It is also...

... creatures commonly found in freshwater ponds, provide a good potential solution to this problem. Planarians...companion resource called “cutting class” which provides more detailed materials and protocol resources...synthetically made RNA (riboprobes) with modified nucleotides (for example, Digoxigenin-labelled Uracil) that...dsRNA feedings. Some of the phenotypes are not evident until after amputation. For example, the dramatic...odf2 gene knockdown results in an observable “sidewinder” phenotype that disrupts wildtype locomotion....dsRNAs into PR-T4P. The Sánchez lab protocol also provides step-by-step instructions for the “the bravest...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ..., CRY2clust, was created by adding a 9-residue peptide to the C-terminus of human codon-optimized  cryptochrome...conditions. With its additional 9 amino acid peptide, the CRY2clust domain enables fast and reversible...original version containing CRY2 without the extra peptide. CRY2clust should prove a useful tool to further...supplied in the media, these bacteria are unable to divide. However, if fueled with caffeine and equipped ...Dronpa flanked a PKA substrate fused to the phosphopeptide binding domain FHA. The FHA1 domain binds to...

...given peptide can easily be tested by generating Level 1 TUs with unique promoters, and the ideal phosphatase...behave in a cell. For unpredictable cases, MoClo provides two answers: first, sets of parts with characterized...for more complex CRISPR experiments (e.g., many guides/parts delivered) in certain model organisms. In...combinations of different candidate genes at once to identify the best cocktail of genes when the relationship...Resources on Addgene.org Addgene’s Modular Cloning Guide EcoFlex MoClo Kit CIDAR MoClo Kit MoClo Plant Parts...

...using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment (Figure 1...recombination event then excises the DNA between the identical loxP or lox511 sites (Figure 1, bottom). Since...only one loxP and lox511 site will remain on either side of the DNA fragment, any additional recombination...genetics, FLEx is currently being used to study a wide variety of in vivo conditional gene rescue experiments... silent until a cell or tissue-specific Cre is provided. Since the FLEx switch ensures Cre-dependent reporter...inverse Orientation), FLEx systems are generally considered "Cre-On" meaning that your gene of interest starts...

... of the DNA with a high frequency of C and G nucleotides). The conversion of cytosine to 5-methylcytosine... to study epigenetics Epigenetic research uses a wide range of molecular biologic techniques to further...enzyme digestion, DNA adenine methyltransferase identification, and bisulfite sequencing. However none of ...consists of one substrate (the histone-derived peptide) which can localise the construct specifically ..., which binds selectively to lysine-methylated peptides. This construct is in the middle of a FRET pair...When methylation occurs at the histone-derived peptide, the methyllysine binding domain causes a structural...

...While “pictures” generated with current 3C methods provide useful information about chromatin interactions...streptavidin IP. Biotinylated dCas9 goes along for the ride throughout all of these steps, until its connection...off-target sites. Long-range chromatin interactions identified with the multiplexed CAPTURE 2.0 largely replicated...replicated interactions previously identified with the original CAPTURE method. Both CAPTURE methods outperformed...both the number of unique chromatin interactions identified and the level of on-target enrichment. 2. Determining...on Your CRISPR Background with Addgene’s CRISPR Guide Page ...

...breaks, in which nucleotide sequences are exchanged between two similar or identical molecules of DNA....homology to the locus of interest. To give you an idea of the process, we’ll walk through an experiment... gene have been cloned into the vector on either side of an antibiotic resistance gene. To avoid selecting...HSV thymidine kinase (HSV-tk) is included just outside one of the homology arms. When we select cells ...creating knockout animals or cell lines, please consider sharing them by depositing with Addgene! Figures...

...fluorescent proteins and luminescence have been widely used in biological research for a long time, chromoproteins... give colors in ambient light. In contrast, the widely used green fluorescent protein (GFP) gives a bright...early 2000s, a handful of chromoproteins were identified in sea anemone (Chan et al., 2006). Why do these... sequencing are more definitive, but visual identification is a good place to start before taking on more...could get around this problem. There is no single “ideal” chromoprotein plasmid that combines the most desirable...is an orange light-absorbing chromoprotein that Hideji Murakoshi’s lab developed as an acceptor for fluorescence...

...method into six general steps and outlined the considerations to be made during each step. Now that you know...take a slightly deeper dive into those steps and provide you with more details to help you plan and optimize.... If your target is already on the extracellular side of the membrane, then this step may not be necessary...binding to any target protein that is still on the inside of the cell. However, assuming you do need to permeabilize... of fixatives or permeabilizers, but how do you decide which ones to use? Here are some tips for making... then optimizing from there is a good option.  Consider your target - if you are studying a membrane protein...

...validated for BiFC (Kodama and Hu, 2012). Having a rough idea of how your PIPs might interact will allow you to...each PIP and empirically test which combination provides the best fluorescent signal. As for linker sequences...experiments. In either case, overexpression should be avoided because it can generate high background fluorescence...critical to the success of BiFC experiments. The ideal control is a mutated variant of one or both of your...that you empirically test a number of mutants to identify those that inhibit BiFC in your cellular system...endogenous PIPs is expressed at increasing levels alongside your BiFC constructs. If your PIPs actually interact...

... Protocol Addgene's Video Antibody Purification Protocol Addgene's Antibody Guide References Trimmer JS...the periplasmic space to be oxidized and form disulfide bonds (Frenzel et. al., 2013). If you’re using...stained, the heavy and light chains become visible alongside your ladder and a protein standard. The density...calculated using imaging software. Any bands on the gel beside the heavy and light chains can also be measured...multiple contaminating bands or a few strong ones, consider increasing the number of washes performed during...

...was in its infancy, but the eye was identified early on as an ideal target. It is an easily accessible ... time advances in human genetics led to the identification of several disease-causing genes, including...However, AdV was abandoned early on due to its considerable immunogenicity and its transient expression....have broad tropism in the eye. AAV-2, the first identified serotype and the one used in Luxturna™, can efficiently...therapies is impressive: clinical trials for choroideremia, X-linked retinitis pigmentosa, achromatopsia...

...an IV injection is an ideal way to deliver AAV to the PNS. It’s a happy accident that AAV.PHP.S targets...barrier - only those that cross the barrier can be identified by PCR later. The PNS presents a different problem...Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nature biotechnology...Additional Resources on the Addgene Blog Important considerations when using AAVs Learn more about using AAV ...Vectors to use with your research. Visit Our AAV Guide Page for info on AAV components, common uses of ...