...join one peptide tag to another peptide tag. Addgene: What is the advantage of peptide tags for building ...locking two peptide tags together. SpyLigase overcomes limitations in the use of peptide tags, which often form...Howarth: Peptide tags can be very generic; you should be able to use the same peptide tag for all sorts ...Natl Acad Sci U S A. 2014 Mar 17. PubMed) Peptide tag forming a rapid covalent bond to a protein, through...form only weak and reversible bonds. Howarth’s team has already demonstrated in their PNAS paper that SpyLigase...discovered the domain. It is small, highly stable and has this amazing chemistry locking the domain together...cancer in particular. They recently introduced the SpyTag/SpyCatcher system, based on a protein isolated ...

...system for protein tagging. We spoke with Mendenhall and Myers about their new CRISPR tagging system, which...www.addgene.org/crispr/tagging. Addgene: What motivated you to adapt CRISPR for use in protein tagging? Myers: Our...epitope tag onto the transcription factors. By the way, you can actually use this for tagging any protein...population of cells with the tagged transcription factor. The FLAG antibody has been well characterized and...factors. Our groups got together to adapt CRISPR for tagging experiments and it’s already looking like it’s ...correctly, the second plasmid inserts a small epitope tag called FLAG at the end of the transcription factor...of advice for people who may want to apply this tagging system to their own work? Mendenhall: We’ve made...

... SpCas9 is also fused to a 3X FLAG tag at its N-terminus. This tag can be used to test for SpCas9 expression...Alternative forms of this vector contain GFP-tagged and un-tagged versions of SpCas9 and further variants ...modified version of CAG promoter that the Zhang lab has tested in a variety of mammalian cell lines. Cas9...effective in certain cell lines. This point mutation has been corrected in V2.0. pSpCas9(BB)-2A-Puro (PX459...that, upstream of the Cbh promoter, this plasmid has a U6 promoter followed by BbsI restriction sites ...

...Twelvetrees Lab took advantage of the self-labeling tags SNAPf and CLIPf. These tags rely on synthetic dye...single- and double-tagged constructs (including "CLAP", combining an N-terminal CLIPf tag and a C-terminal...studying mEPSPs has been possible with conventional electrophysiological methods, it has remained difficult...dicots Self-labeling tags for single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors...2024.02.11.579803).  CLIP, CLAP, SNAP! Self-labeling tags for single-molecule biophysics By Mike Lacy In order...C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression and labeling approach ... The team deposited an array of constructs for tagging the kinesins KIF5A and KIF5B as well as backbones...

...protein available – you can always add an epitope tag or a fusion protein. Mammalian vectors One of our...to be a more robust expression system and has its advantages; however, remember that you will need accessory...pBAD LIC cloning vector (8A)) and have an epitope tag (6xHis or GST) to affinity purify the expressed protein...popular studies involve using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following...& Other Plasmid Protocols List of common epitope tags ...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...

...interactions. The Roux lab has deposited HA tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal...for an individual tagging event. Commonly used sequences, including fluorescent tags, a floxed Cbr-unc...proteins are tagged with Protein A-Renilla luciferase and the “prey” proteins are tagged with Firefly ...organisms has limited routine use of this technology. To streamline CRISPR/Cas9-based genetic tag insertion...generation lentiviral transfer vector. Each gene has been FLAG-tagged for convenient experimental use. The plasmids...with SpyTag/SpyCatcher and SnoopTag/SnoopCatcher The development of recombinant DNA technology has allowed...119 selectable marker, and “connectors” that link tags to the targeted gene are supplied to the reaction...

...of interest is purified using an affinity tag. Additional tags may be used to improve protein stability...researcher to test multiple tags for one protein quickly and easily. These tagged plasmids also include cleavage...remove the tag post-purification. A sample of pCri constructs: pCri-1a, -4a and -9a. 6xHis tags enable affinity...remove these tags from the purified protein. For more detailed information on these and other tags used in ...integral-membrane protein constructs) protein tag. MISTIC drags tagged proteins into the membrane and may improve...which includes plasmids with different varieties of tags, both N- and C-terminal. Each set of plasmids uses...solubility and stability. TRX and signal peptide tags help proteins with disulfide bonds fold properly...

...protein tags can be used to facilitate the visualization of specific proteins in cells. Tagged protein...containing AHA to a fluorescent alkyne tag. The newly synthesized and tagged proteins can then be observed with...the knock-in of fluorescent tags into the gene locus of interest. An advantage of this alternative is the...here, there is the question of whether or not the tags influence behavior. A conference and a breakthrough... known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich et al. Nature Neuroscience, ...protein of interest that also had incorporated the AHA tag after pulse labeling of a determined period of time...used for cell labeling Check out other protein tagging strategies Additional Resources on Addgene.org...

...Nature Methods Find the anti-CRISPR plasmids Tagging human transcription factors with GFP Kevin White...lab recently deposited a set of CRISPR plasmids to tag human transcription factors with GFP. As part of ...more about ENCODE Find the transcription factor tagging plasmids If you have a new CRISPR tool you’ve...dual-nuclease Cas9-Cas9 chimeras Scot Wolfe’s lab has developed dual-nuclease Cas9-Cas9 chimeras with higher...been many Cas nucleases developed for plants, it has been difficult to compare the performance of different...recently discovered in 2017. Now Dominik Niopek’s lab has adapted anti-CRISPR proteins for optogenetic control...

...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...

...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...

... 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...

...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...

... 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...

...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 ...

...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...

...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,...