...purification tag blog!).   Applications of epitope tags   The tags A number of epitope tags exist...guidance on designing a GFP tag Addgene has you covered!   Potential tag issues Tags have a lot of major benefits...hemagglutinin. The tag has two negatively charged amino acids for an overall negative charge. This tag has been used...Addgene has you covered with monoclonal antibodies to Myc for all of these species. HA The HA tag is a ...fix your antibody woes.   What are epitope tags? Epitope tags are short peptides introduced at the N or...system. Tagging can also aid in protein purification! In this blog, though, we are focusing on tagging for...amino acids on the C terminal of the tag. Of the most common tags, FLAG is the most charged, with 5 negatively...

...feature provides HA frankenbody the ability to bind nascent HA tagged proteins.  Lastly, the HA frankenbody...proteins and tags including mCherry, HaloTag and SNAP-tag for multi-color imaging. Find the HA frankenbody...observed colocalization of GFP tagged HA frankenbody with an mCherry, 4xHA tagged histone protein (H2B) in ...mitochondrial protein tagged with spaghetti monster HA (smHA) which contains a 10xHA tag. These experiments... towards a HA epitope from the CDRs.  To visualize proteins of interest tagged with the HA epitope, the... the HA frankenbody? Another large advantage of the HA frankenbody is that it remains bound to HA epitopes...localization of single HA-tagged molecules over time.  The Stasevich lab also proved that HA frankenbody can ...

...innocuous tags” (inntags) less likely to alter a protein’s properties. Why do we need new epitope tags? Short... looking to a tag a protein, inntags IT5 and IT6 are a great place to start. These tags are easy to use... about other Protein Tags in our Plasmids 101 Series. Get the Inntag Plasmids Tag endogenous proteins ...major caveat - a tag may interfere with protein localization and/or function, so each tagged protein must ...the effects of GFP fusions to inntags or traditional FLAG, HA, and Myc tags. Impressively, IT6-GFP did not...Short epitope tags like FLAG, HA, and Myc can adopt a number of different conformations and thus potentially...with IT5 and IT6 fusions. In contrast, Flag and HA tags had especially detrimental effects on cell size...

...affinity tag is the polyhistidine (His) tag. Small and unlikely to affect function, His-tagged proteins...greatly alter the size of the tag.   Table 1: Common protein tags Tag Epitope Mass (kDa) Function Notes...Combo and cleavage tags Frequently, a single tag is not enough. What if you need one tag to increase solubility...to all tags, but rather a quick overview of why scientists use tags, with a few time-tested tags and fusion...to fuse an affinity tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses...keep your protein soluble.  Tags for affinity and purification An affinity tag, generally a relatively small...usually using a Ni2+ column. Like other affinity tags, a His tag can be fused to either the N- or C-terminus...

...RANbody is detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties...Spaghetti Monsters are highly antigenic tags that have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP...Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B Myc Tagged RFP, H2A2B Chicken IgY (Y) Labeled...discussed in more detail below. The HA tag can be stained for with an anti-HA antibody and serves as a backup... Spaghetti Monster tagged RANbodies can be detected either with a labeled anti- HA or -MYC antibody or...Gelsolin Spaghetti Monster: HA (H) or Myc (M) versions Labeled anti-HA or -Myc antibody Staining cultured...lot-to-lot variability in activity. No Genetic Tagging - Since antibodies are produced in animals instead...

...transgene expression. Which tag should I use? When deciding on the appropriate tag, the experimental design...DREADD to the plasma membrane. The haemagglutinin (HA) tag is a small 9 amino acid epitope of the haemagglutinin... big difference. Replacing the mCherry with the HA tag resulted in abundant expression of hM4Di in plasma...fluorescence is not needed, the use of HA and other short epitope tags (Kimple et al., 2013) could help improve...to reveal the hM4Di coupled to mCherry (top) or HA tag (bottom). The images show examples of dendrites...to the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...our studies suggest that non-fluorescent tags may be advantageous over fluorescent proteins when the fluorescence...

...degron tags. What is a degron tag and why should I use one?   Degron tags are a class of protein tag which...amenable to being tagged, e.g. tagging some proteins can affect their function, the SMASh tag self-cleaves ...). Choosing a degron tag Are all tags created equal? No! Choosing the ‘right’ tag does depend a lot on...Protein Tags Plasmids for Endogenous Gene Tagging in Human Cells Antibodies 101: Epitope Tags  ...) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation tag (dTAG) system functions...degradation. Like several other tags described here, the dTAG uses a small molecule, a tag, and the endogenous cellular...machinery to function. The dTAG is the smallest tag reviewed here, in regard to total tag size, which can also...

...strep-tag and, more recently, OLLAS (Escherichia coli OmpF linker and mouse langerin) and Sun Tag (Viswanathan...antibodies can have low affinity for these tags and even multimeric tags are frequently insufficient for detection...FP and epitope tags, researchers in Loren Looger’s lab have developed new molecular tags combining the ...antibody recognition of epitope tags. Thus they have created antigenic protein tags called “spaghetti monster...experiments with tag-specific antibodies. The most famous ones are influenza hemagglutinin (HA), myelocytomatosis...With the limitations of epitope tags and fluorescent proteins, it has been crucial to develop new tracers...provided better labels than fusions to 3 or more HA tags. 4. High-resolutions microscopy As indicated above...

...T2A-TurboGFP-PEST), and small epitope tags (HA, Myc, Strep tag II, AviTag, HaloTag, SpyTag). The Foerstemann lab developed... scarless tag insertion.   In addition to the obvious advantages SapTrap offers for tagging a single genetic...between the tag and homology arms). Digestion of the donor plasmids with SapI allows the tag, selectable... inserting a tag at multiple target sites. SapTrap vectors can be easily modified to tag hundreds of genes...insertion tagging), developed by the Hornung Lab, allows users to easily create C-terminal tag fusions ...endogenous protein tagging, please visit our website CRISPR/Cas Plasmids - Protein Tagging. Beyond simply...expression levels. CRISPR tagging systems offer users an easy and efficient way to tag endogenous proteins ...

... His-tagged protein purification work flow by nickel column.    Affinity tags vs. other tags There...different types of tags, including epitope tags (FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry...GST antibody? Addgene has you covered! His His tags, often called polyhistidine tags, typically include ...planning, affinity tags can make protein purification a cinch. Types of affinity tags, the proteins they...are all reviewed here. What are affinity tags? Affinity tags are used primarily as protein purification... common tags available. Maltose binding protein Maltose binding protein (MBP) is a 45 kDa tag which can.... The MBP tag, although large, can actually increase expression and solubility of the tagged protein (...

..., modular plasmids, combining fluorescent tags, epitope tags, protease sites, and selection markers. These...fluorophore tagging vectors for your single or multi-color imaging experiments. Interested in epitope tags? Others...have genome tagging systems enabled you to advance your research? Do you have a favorite tagging system that...at the University of Utah, has built a complete collection of yeast tagging modules with each possible...linker-(epitope tag)-ADH1 terminator-(selection marker). Additionally, Dr. Formosa has deposited six plasmids...Researchers have long taken advantage of this natural process to integrate protein tags into the genomes of S...especially useful in protein complex studies where tagging of multiple protein products is desired, as multiple...

...the Myc tag has become one of the most commonly used protein tags - a quick search for ‘Myc tag’ in the...detecting protein tags.  And when you think of protein tags, what are the first tags that come to mind...guess that the Myc-tag is one of them (and not just because of the title!) The Myc-tag is a string of 10...channelrhodopsins. HcKCR1 has a maximum absorption of 540 nm and two-photon excitation around 1,040 nm, has larger photocurrents...Improved voltage indicator: JEDI-2P Novel class of light-gated potassium channelrhodopsins Myc-tag antibody...https://doi.org/10.1101/2021.09.17.460684.)   Myc-tag antibody anti-c-Myc [9E10] now available! by: Ashley... 3: Image of Addgene’s western blot for the myc-tagged protein GFP-smFP-myc expressed from Plasmid 98926...

...epitope (GKPIPNPLLGLDST) has been used as a tag since 1992 (Hanke et al., 1992), and has proven itself a versatile...Epitope tag antibody collection grows By Ashley Waldron We are excited to highlight the Anti-V5 tag antibody...immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen. J Gen Virol., ...Figure 3: Expression of fluorophore-tagged GOLG-4 through eight stages of embryonic development. Representative...find in this post: Anti-V5 and other new epitope tag antibodies from IPI New tool for structural studies...studies of E3 ligase cereblon Optimized mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene...Innovation (IPI), part of a new collection of epitope tag antibodies available through Addgene. Anti-V5 [IPI-SV5...

... on the polyhistidine tag. What is the polyhistidine tag? The polyhistidine tag is a string of 6 or more...polyhistidine tag, alternative fusion tags were used, such as S. aureus’ Protein A (Moks 1987). These tags, however...conditions.   Find polyhistidine tag plasmids here! Where should I place the tag and how many do I need? When...containing a polyhistidine tag, you’ll need to decide whether to add the tag to the N- or C-terminus and...requires the tag to be accessible to the metal ions once the protein is folded. Sometimes a tag placed on... however, tag placement can affect protein function. B, The string of histidines in the tag is typically... small size of the polyhistidine tag minimizes the chance of the tag disrupting normal protein function...

...epitopes tags such as HA and MYC in vivo. The authors found that fusions to one particular tag, the IT6... for epitope tagging: Inntags When fused to your protein of interest (POI), epitope tags give you the...production of a new series of epitope tags, inntags (innocuous tags), that they believe will have fewer ...expressed either without a tag or as multiple fusions with different N- and C-terminal tags. Additionally, by ...expression of fusions between the POI and a TAP tag (a tag with two epitopes that can be used to sequentially...two dimeric Broccoli tags (2xdBroccoli) to further enhance fluorescence of the tagged RNA. Bacterial and...containing a variety of N- and C- terminal tags. The Santalucía Lab has developed a cloning toolkit based on...

...is to tag the protein of interest with a small biochemical “epitope tag” such that these tagged proteins... the tagging efficiency in the transfected cells, we count the number of tag-positive cells (tag ICC) ...endogenous gene tagging in human cells Plasmids 101: Protein Tags Fluorescent tagging of endogenous genes...crRNA and/or try out an alternative tag (3xFLAG, Myc, V5, or HA). Tag-specific PCR genotyping can tell if...each with a different tagged protein of interest, and a universal all-purpose “tag antibody” can be used...projects that require epitope tagging of a large cohort of genes (e.g. tagging genes representing a protein...pipeline for epitope tagging. Using our 96-format pipeline, we can routinely achieve 5-30% tag knock-in efficiency...

... the best outcomes. The go-to in vivo option AAV has become the preferred delivery method for in vivo ... other viral vector delivery methods. Lentivirus has limited packaging options, with most lentiviral vectors...possibilities when designing screens. Each serotype has its own tropism, or a preference to infect specific...BioRender.com. Beyond the identified serotypes, AAV also has psuedotypes. Psuedotyping is the process of making...Lentiviruses have capacities of 8–12 kb, while AAV only has approximately 4.7 kb — which means that when using...the genetic landscape of tissue-specific diseases has grown exponentially, aided by advancements in in ...ones. In those cases, AAV vector screens may be AAVantageous!       In vivo, in vitro, ex vivo CRISPR screens...

...3xFLAG and biotin tags, use of tag systems including Protein A, 1xFLAG, 2xAM, and HA have been reported...2013). However, the fusion of dCas9 with a protein tag allows for the isolation of a genomic region of interest...cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting their genomic...Immunoprecipitation, using an antibody targeting the 3xFLAG tag, allowed these researchers to isolate dCas9 with ...chromatin or DNA fragment (Fujita et al., 2016). 3xFLAG-tag fused to dCas9, along with a biotinylated gRNA, were...were successfully used in this "in vitro" form. Any tag system can be used to isolate genomic regions of ...This system utilizes dCas9 fused to a biotinylation tag expressed on a plasmid, allowing for in vivo biotinylation...

... small epitope tag (FLAG is eight amino acids, HA is nine), multiple repeats of the tag can recruit multiple...SNAP-tag, and HaloTag fusions, while the anti-HA frankenbodies include mEGFP, mCherry, and HaloTag. ...blotting, and more. Given the popularity of FLAG and HA tags, you may not even need to clone new constructs ...b) Schematic of anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of...cassette is advantageous to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally .... The proteins, which are purified via histidine tags, are optimized for SDS-PAGE gels and can be detected...targets by: Mike Lacy Adding to their set of anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and...

...introduced the Myc tag to the world, it has become one of the most commonly used tags in molecular biology...instance, the commonly used Myc-tag. Who first started using it in protein tagging experiments? Why Myc? When...explore… the life and times of the myc tag.  Origins of protein tags For about as long as the disciplines...was within the tagged protein rather than at the C-terminus. These features made this tag easier to use...“equi-kli-seed-le”). (3) Tag all the things!   Myc1-9E10 and the Myc-tag today Since Munro and Pelham... amino acids, meaning the tag could be quite small. However, substance P has an amide group added to its...high quality antibodies available - aka peptide tagging.  Key to this strategy was the existence of high...