...C-terminal fusion of a tetracysteine tag. In addition, since these tags are so small, one can insert them... lure the ribosome into inserting a fluorophore-tagged amino acid at a stop codon (often amber i.e. UAG...termination at stop codons, expression levels of these tagged proteins are often very low. In addition, a major...which can be carried out using Addgene plasmids. His-tag labeling reagents You may already have a FRET acceptor-binding...: you just don’t know it yet! The poly-histidine tag often used for protein purification is a reliable...for protein purification: the NTA binds to the His tag on your protein and the coupled Cy fluorophore acts...well as by extending the length of the histidine tag from 6 to 10 residues. While these Cy/NTA conjugates...

...nucleus to cell membrane, this collection has fluorescently tagged versions of many proteins known to localize...spectra. A comprehensive list of cell markers all tagged with mEmerald. Plasmids to monitor protein-protein...available targets since the Davidson lab always tagged new proteins with mEmerald first, Baird says. From...story about his life’s work and the stage IV lung cancer that has now prompted him to deposit the tools...Michael Davidson’s lab at Florida State University has been the go-to place. In 2012, his National High ...backbones alone,” said Addgene’s Lianna Swanson, who has been working with members of Davidson's lab to coordinate...coordinate the impressively big deposit. “He has every fluorescent protein under the sun, from the standard...

...gene expression patterns. The lab has also used CellTags for tagging two cell populations in a competitive...the single-cell level with CellTag CellTagging is a lentiviral based tagging method that labels cells with...Each cell is labeled with 3-4 tags to increase the diversity of the CellTag library. The fixed region acts...library-specific identifier and allows cells to be tagged with different CellTag libraries at multiple timepoints during...inefficient. Cells were sequentially tagged with different CellTag libraries at three time points during...reprogramming outcomes. For example, cells with shared CellTagD13 tags often had similar gene expression pattern changes...to use CellTag, you can find the CellTag libraries at Addgene. The Morris Lab's website also has several...

...blog has you covered. Happy voltage sensing!   Figure 3: Components of the types of voltage indicators...approaches. Functionally, the voltage sensitivity of the available VSDs has been successful even at the ... Several adaptations of this method have been to tag, cage, uncage, and trap the chemical fluor with the...single neuron level in vitro. In vivo monitoring has also been achieved with these sensors, but further...research tools for studying voltage within a cell are genetically encoded voltage indicators (GEVIs) and chemigenetic...Types of Voltage Indicators GEVIs can be divided up structurally into 2 main groups: voltage sensitive...occurring voltage indicator. To give an example, a genetically encoded phosphatase (voltage sensing) hydrolyzes...

...anti-FLAG tag antibodies By Ashley Waldron Of all the epitope tags we know and love, the FLAG tag (amino ...purify or image a recombinant protein, the FLAG tag has proven itself a good option for essentially all...Endothelial-specific AAV delivery Recombinant anti-FLAG tag antibodies Peptide-assisted genome editing for ...collection includes over 10,000 plasmids with a FLAG tag (!), but did you know that we distribute ready-to-use...FIQ or mStayGold (Ando et al., 2023). This variant has similar photostability and brightness compared to... of AAV9-X1.1 to that of AAV1. This viral vector has brain endothelial cell-specific tropism, with slightly...western blots, though the original parent clone (M2) has been widely used in a variety of applications. Find...

... a unique tag into a genomic site of choice via the mutational abilities of PolI3m. This tag would allow...used. EvolvR: key components The EvolvR system has two key components: the error prone PolI3M that’s...BioRender.com. The initial version of EvolvR has an editing window of ~17 nt from the nick site, which...directed mutagenesis to generate specific mutations in your plasmids Use REPLACER Mutagenesis to mutate...Mutagenesis is a tool that both evolution and molecular biologists use to tinker with DNA. Making changes...region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley...potential applications. Challenges of existing mutagenesis methods Several continuous diversification methods...

... with containing chemical tags such as the SNAP-tag, Halo-tag, and CLIP-tag (Kohl et al., 2014) (Addgene... et al. "Ultrafast tissue staining with chemical tags." Proceedings of the National Academy of Sciences111.36... circuits with fluorescent proteins and chemical tags." eLife 7 (2018): e40350. PubMed PMID: 30454553.... an assistant professor at Kyushu University. He has a particular interest in the development of sensory...Imai is a professor at Kyushu University. His lab has been studying functional and anatomical aspects of...

...DNA for use with virus-mediated delivery has many advantages for research. Viruses facilitate the delivery...Kb 80-130 Dividing Yes Moderate * Recombinant AAV has a low frequency of target cell genome integration...

...to their N-terminal His14-bdNEDD8 tag. After several washes, this tag can be cleaved by the bdNEDP1 protease...sacrifice and precludes genetic engineering of a tag or fusion with a reporter enzyme. For multicolor ...and decreases the distance between a fluorescent tag and the target antigen to about 2 nm, which can lead...antibody can be “labeled” by incubating it with a tagged nanobody. After this pre-incubation step, the primary...Label the Nanobodies: Purified nanobodies are then tagged with a fluorescent dye or HRP via maleimide-labeling...Control Protein Activity Learn more about protein tags Read about E. coli strains for protein expression... a primary antibody. B) Each arm of the antibody has an antigen binding site for a total of 2 binding ...

...disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind...REPLACR-mutagenesis could save busy scientists a lot of time. Let us know how REPLACR-mutagenesis works ...Comparison of REPLACR-mutagenesis with other common methods for site directed mutagenesis   References 1. ...al. (2016). “REPLACR-mutagenesis: a one-step method for site-directed mutagenesis by recombineering.” ...Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular...vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering... Plasmids After PCR). Current site directed mutagenesis methods Current SDM methods rely on in vitro ...

...contain a variety of N- and C-terminal tags (including His, S-tag, OneStrep, CBP, Trx, GST, Halo, MBP, ...promoter, N-terminal tag, coding sequence of the gene of interest, C-terminal tag, plant terminator, and...Ting’s lab has enabled progress on both fronts – as a genetic electron microscopy (EM) tag providing superior...of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated the use of the SpyLigase...vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular...Building off their SpyTag/SpyCatcher system for protein-peptide locking, Mark Howarth’s lab has developed a ... superior subcellular localization of tagged proteins compared to traditional fluorescence microscopy,...

...combines components of SAM and SunTag activators, fusing the epitope tag of SunTag with the P65-HSF activation...SAM. This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou..., Qi LS, Weissman JS, Vale RD (2014) A Protein-Tagging System for Signal Amplification in Gene Expression... for CRISPR activation, research in the area has continued to develop. These newer methods have not yet... of rearranging chromatin structure. This method has shown higher activation levels than SAM in some loci...popular activation methods (VPR and SunTag) (Chavez et al., 2016). SunTag Description Rather than using a ...activation levels are similar to that of SunTag. An advantage to this method compared to other notable...

...KB Fujii iChIP/enChIP to purify genomic DNA FLAG tagged dCas9 PDF, 110 KB Goldstein Nematode: gRNA design...PDF, 109 KB Mendenhall and Myers Mammalian: FLAG tagging endogenous proteins pFETCh_Donor ; additional HDR...cloning in gRNA: pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) PDF, 49 KB Vosshall and Matthews (Link opens...

... cells. Flexible tagging: Choose untagged or add a versatile N- or C-terminal HA tag. Selectable and stable...EGFP into pAG Lenti CMV N-HA Puro (Addgene #236079) to create pAG Lenti CMV HA-EGFP Puro (Addgene #236081...breeze. Modular by design: Key elements (promoter, tag, polyA site) are flanked by unique restriction sites...plasmid set, our bacterial plasmid set for expressing tagged proteins, and stay tuned for mammalian expression...streptavidin immunoprecipitation (IP), and tandem mass tag (TMT) mass spectrometry (MS) in HCT116 cells (colorectal...by the Loren Looger and Kaspar Podgorski Labs now has a new version: iGluSnFR4, developed by the GENIE ...we're excited to share our very own set of empty, HA-, and EGFP-expressing lentiviral backbones designed...

...Advanced Microscopy (University of Amsterdam). Tagging a protein of interest with a fluorescent protein...still block the function or activity of the tagged protein because of its size. This type of interference... is known as “stickiness.” Since the fluorescent tag should operate as an inert light emitting module,...successful insertion of a fluorescent protein are the tagging of Ccd42 (Bendezú et al., 2015) and the alpha subunit...most notably CRISPR/Cas mediated HDR) enables the tagging of endogenous genes close to native expression ...protein variant, and where to add the fluorescent tag. These choices will determine how well the fusion...reflects the function of the native protein that is tagged. The localization of the fusion protein can be ...

...extracts (Gagoski et al 2015). He has also constructed a library of eGFP-tagged human ORF clones to allow testing...-terminal eGFP tag carried on all constructs. The Cell-Free Protein Expression Kit has been used to produced...additionally contain various combinations of protein tags (His, Avi, and GFP) as well as protease sites to...researcher to quickly generate N- and C-terminal epitope tag fusions to a protein of interest. The plasmids used...folding and/or protease sites to remove the epitope tags. Different plasmids in the kit can be used to purify...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...

...pAAV-TK-Acceptor has a conventional heterologous promoter-driven TK-neoR gene. To modify these tagging vectors ...their lab include using AAV to introduce epitope tags into the endogenous alleles of the p53 and PTEN ...allele of the STAG2 tumor suppressor in human cells (Kim et al, 2016). The Waldman lab has designed their...their AAV tagging vectors (pAAV-SEPT-Acceptor and pAAV-TK-Acceptor) so that they can be easily adapted ... introduction of heterozygous endogenous epitope tags, or the introduction of dominant mutations of a ...: PMC373311. 3. Kim, Jung-Sik, et al. "Epitope tagging of endogenous genes in diverse human cell lines... at the Georgetown University School of Medicine has used this gene editing system frequently over the...

...Tet-on transgene vector. Degron tags Tagging relevant proteins with a degron tag can provide inducible protein...systems work by tagging the endogenously or exogenously expressed protein with the relevant tag and then introducing...ligand to mark the tag and whatever it is fused to for degradation. Since the tagged protein of interest... are akin to degron tags, just with a flipped mechanism. When a protein is tagged with a DD, it is unstable...auxin-inducible degron (AID), and degradation tag (dTAG). These control systems function at the post-translational...Check out Addgene's Lentiviral dTAG cloning vector and SMASh degron tagging vector! Destabilizing domains...quickly recover once the ligand is removed. Degron tag systems include small molecule assisted shut off ...

... well as additional useful tags, including 3xFLAG epitopes, TEV and BioTag, which can be used in protein... protein effectivity), and a fluorescent protein tag for visualization (Fig. 1b). While the probes target...ultimately leading to the degradation of the AID-tagged protein. To quantify protein degradation the Ward...44 amino acids auxin inducible degron sequence) tagged proteins. Degradation of your protein interest ...Gibson and Saptrap cloning systems to generate AID* tagged CRISPR knock-in repair constructs. These repair...repair constructs can be used to integrate the tagged AID* into genomic sites of interests via CRISPR techniques...Cecchetelli The auxin inducible degron (AID) system has been extensively used in C. elegans since 2015 to...

...overview). Until recently, the approach has been hampered by the need to tag each protein of interest and, for... any transgenic zebrafish line expressing a GFP-tagged version of a protein of interest. The binding of...labeling proteins in close proximity to the GFP-tagged protein (albeit slightly offset due to the nanobody-GFP...   By Ashley Waldron Proximity-dependent labeling has become a powerful approach for proteomic mapping ...M-CREATE) by: Brian O'Neill  The Viviana Gradinaru Lab has previously developed the multiplexed version of the...sequence knock-ins by: Lucie Wilson Le Cong’s lab has developed a method using dCas9-SSAP to knock-in long... methods for large inserts. However, this system has a much lower error rate. For a one kilobase insertion...