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We narrowed to 527 results for: HA tag

Showing: 61 - 90 of 527 results

  1. Viral Vectors 101: Voltage Indicators

    Type
    Blog Post
    ...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...

  2. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    ...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...

  3. Single-cell tracking of lineage and identity with CellTag

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    Blog Post
    ...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...

  4. The Michael Davidson Collection: One-Stop Shop for Fluorescent Proteins

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    Blog Post
    ...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...

  5. Targeted Mutagenesis with EvolvR

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    Blog Post
    ... 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...

  6. Hot Plasmids February 2024

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    Blog Post
    ...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...

  7. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

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    Blog Post
    ... 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...

  8. Plasmids 101: Secondary Nanobody Toolbox

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    Blog Post
    ...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 ...

  9. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    ...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 ...

  10. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

    Type
    Blog Post
    ...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...

  11. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...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,...

  12. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ...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 ...

  13. Hot Plasmids: Spring 2025

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    Blog Post
    ... 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...

  14. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ...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...

  15. Plasmids 101: The protein expression toolbox

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    Blog Post
    ...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 ...

  16. Adeno-associated Viruses (AAVs) for Genome Editing

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    Blog Post
    ...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...

  17. CRISPR References and Information

    Type
    Collection
    ...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...

  18. Hot Plasmids and Viral Preps - March 2021

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    Blog Post
    ... 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...

  19. Cancer Research Plasmids and Resources

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    Collection
    ...Research Cancer Research Plasmids and Resources Addgene has a large variety of resources for use in cancer signaling...vectors for in vitro and in vivo research. TP53 Mutagenesis Library : Pooled library from the Hahn and Root...

  20. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

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    Blog Post
    ...these self-labeling proteins are SNAP-tag® and HaloTag®. These tags consist of a genetically encoded enzyme...several advantages to using these self-labeling proteins over traditional fluorescent protein tags, such...endogenous target, and thus no genetically encoded tag is required. These dyes can also be attached to an...ligands have more flexibility due to the affinity tag they typically bind to. Stability of fluorescence...experimental labeling. Size is also a factor. Bulkier tags, such as GFP, may interfere with protein expression...fluorescent experiments, such as using fluorescently tagged ligands or intermolecular FRET for ligand-receptor...should possess high specificity to ensure accurate tagging. In Fluorescence Resonance Energy Transfer (FRET...

  21. SunTag and Fluorescent Imaging

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    Blog Post
    ...superfolder GFP (sfGFP) with the small solubility tag GB1. In SunTag nomenclature throughout the rest of this ...tracking Caveats of SunTag Very large size: a 24x scaffold fully occupied with GFP has a molecular weight...References 1. Tanenbaum, Marvin E., et al. “A protein-tagging system for signal amplification in gene expression...concerns are greater with SunTag. v1 SunTag exhibits some scaffold aggregation (v4 Suntag does not.) It’s up...may be toxic to the cell. Here comes the SunTag How does SunTag fix these problems? Instead of directly...power of SunTag for single molecule imaging, finding that plasma membrane-targeted CAAX-SunTag was 18-fold...traditional FP fusion, a SunTag fusion could be used as well. Pros of SunTag compared to traditional ...

  22. Hot Plasmids - September 2019 - CRISPR, Auxin Inducible Degradation, MerMAID Channelrhodopsins, and Genome Looping

    Type
    Blog Post
    ... and other tags (Yesbolatova et al., 2019) using CRISPR. This toolkit makes it easy to tag your protein...the Kanemaki lab has created a new toolkit of plasmids for the N- or C- terminal tagging of proteins with...here.        New toolkit for N or C terminal tagging of proteins for degradation using AID Article contributed...specific ubiquitin ligase pathway needed for AID. By tagging proteins with a 7kD degron called mAID and expressing...previously developed a CRISPR/Cas9 based method to tag endogenous proteins in HCT116 cells with mAID. Using...inhibitor, sufficiently suppresses basal degradation of tagged proteins, a common problem of the AID technology...technology (Yesbolatova et al., 2019). Find the mAID tagging toolkit at Addgene Yesbolatova et al., Methods,...

  23. Hot Plasmids - May 2022

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    Blog Post
    ...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...

  24. Antibodies 101: Selecting the Right Antibody

    Type
    Blog Post
    ...antibodies against the same target; tagged protein strategies that express a tagged version of the antigen and ...An antibody that has been cited thousands of times may be a safer bet than one that has never been used...compare staining of the antibody to that of the anti-tag antibody; and immunoprecipitation-mass spectrometry...work. Whenever possible, choose an antibody that has been validated in your specific application. Consider...cross-reacts with. Ideally you will find an antibody that has been validated in the species your protein of interest.... Talk with your peers. If someone in your field has used the antibody before you will not only have peace...Uhlen, 2016). Be sure that the antibody you choose has been validated using one or more of the proposed ...

  25. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ... size distribution and concentration of ApoB-LPs has been shown to have a strong correlation with cardiovascular...indicators difficult to work with.  The Gottschalk lab has surveyed a range of microbial rhodopsin GEVIs in ...information. Such voltage imaging can be performed in intact animals and offers many advantages over traditional...found several voltage biosensors and conditions that can be used to monitor real time voltage changes in ...2019. PubMed PMID: 31451697. Genetically encoded voltage indicators for use in worms Article contributed...contributed by Brook Pyhtila Genetically encoded voltage indicators (GEVIs) change fluorescence in real time when...influence the rhodopsin properties. Find these voltage biosensors at Addgene. Hashemi et al., PNAS. 2019...

  26. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

    Type
    Blog Post
    ...cell surface proteins with a biotin tagged LPETG peptide. Biotin tagged proteins can then be purified using...kD TurboID, which has 15 mutations compared to BirA and the 28kD miniTurbo, which has the N-terminus domain...easily purify and identify any proteins that are tagged with biotin using avidin coated purification techniques...engineered ascorbate peroxidase (APEX), used as a genetic tag for electron microscopy, could be used for efficient...living cells via spatially restricted enzymatic tagging." Science 339.6125 (2013): 1328-1331. PubMed PMID...proteins in minutes rather than hours like BioID. APEX has allowed scientists to identify the proteomic map ...BioID was developed in 2012, proximity labelling has become a powerful technique for studying protein-...

  27. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ...When your gene of interest has a BbvCI site(s). If so, the nicking mutagenesis protocol will still work ...instances where it does. 1) When the backbone vector has multiple BbvCI sites. If these sites have opposite... as template for a site-directed mutagenesis PCR that uses mutagenic primers to introduce all desired ...effects. One-pot saturation mutagenesis The one-pot saturation mutagenesis technique described in Wrenbeck... single mutagenic primer pair to generate an individual mutant, this technique uses mutagenic primers ...saturation mutagenesis with the same protocol. What do you think about one pot saturation mutagenesis? How .... “Random Mutagenesis by Error-Prone PCR”. Methods in Molecular Biology In Vitro Mutagenesis Protocols...

  28. Using Phosphoserine to Study Protein Phosphorylation

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    Blog Post
    ...response to the presence of TAG sequences in ORFs. In this bacterial strain, TAG is an ‘open’ codon, which...ΔA. Because the phosphorserine OTS recognizes the TAG amber stop codon, using the OTS in an unmodified ... recoded to eliminate all 321 amber stop codons (TAG), replacing them with TAA (7). This modification ...through the production of full-length protein (the TAG codon should encode phosphoserine instead of a stop...protein. Up to 80% p-Ser incorporation into E17TAG GFP has been achieved with the Rinehart system (6);...can run samples on SDS-PAGE gels containing Phos-Tag, a small molecule that binds specifically to phosphate...SepOTSl can encode a non-phosphorylated serine at the TAG codon of the same expression vector used for phosphoserine...

  29. Antibodies 101: Immunoprecipitation

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    Blog Post
    ...appealing, but the disadvantages must be carefully considered before using. For example, the tag may affect the... epitope such as c-Myc, GFP or V5 can be used to tag the protein of interest on the C- or N-terminus. ...called a pull-down assay, an antibody against the tag is used to isolate the protein of interest. The wide...interfere with protein interactions of interest. Tagged proteins are typically expressed at much higher...proteins. Data obtained from experiments using a tagged protein may not be translatable to the endogenous...agents. Directly conjugating the primary antibody has the additional benefit of permanent linkage. This...steps following the incubation step. The wash buffer has an optimal pH and ionic strength that breaks weak...

  30. Live and Let Dye: Self-Labeling Protein Tags

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    Blog Post
    ...Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag. Biochemistry, 60(33), 2560–2575...genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag systems Tag Size...self-labeling protein tags. The basics These tags most often consist of an enzyme that has been mutated to ...than short peptide tags like tetracysteine (Jing & Cornish, 2011). HaloTag and SNAP-tag are the most popular...application, SNAP-tag, CLIP-tag (both 182 aa), and eDHFR (159 aa) are smaller than HaloTag (295 aa) – for...resorufin). SNAP-tag: In 2003, Kai Johnsson’s lab developed a self-labeling fusion tag based on the human...later became known as SNAP-tag. There is also a fast-reacting version of SNAP-tag called SNAPf. eDHFR: The...
Showing: 61 - 90 of 527 results