...transgene expression. Which tag should I use? When deciding on the appropriate tag, the experimental design...fluorescence or immunohistochemistry methods, these proteins are routinely fused to ‘tag’ or ‘label’ proteins...our studies suggest that non-fluorescent tags may be advantageous over fluorescent proteins when the fluorescence...detection. Fluorescent proteins are most widely used tags because they emit endogenous fluorescence that allow...protein (GFP) and mCherry, two of the most common tags currently used, are approximately 240 amino acids...most of them were still cytoplasmic. Use of ‘small’ tags to identify proteins Given the limited expression...monkey neurons, we next hypothesized that a different tag fused to the hM4Di might improve the transport of...

... 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 ...epitopes tags such as HA and MYC in vivo. The authors found that fusions to one particular tag, the IT6...expression of fusions between the POI and a TAP tag (a tag with two epitopes that can be used to sequentially...the POI-tag fusion from a plasmid or from a random genomic location than to express the tagged protein...two dimeric Broccoli tags (2xdBroccoli) to further enhance fluorescence of the tagged RNA. Bacterial and...

...techniques researchers are using. The detailed results of his survey can be found in the full post but TLDR: many...optimize and troubleshoot the system. Site Directed Mutagenesis by PCR & Single Base Editing with CRISPR For ...technology may make some uses of Site Directed Mutagenesis by PCR obsolete! Honorable mentions Of course... in chronological order here: Site Directed Mutagenesis by PCR Google Forums Round Up: First Impressions...

...school, I came across Joseph Campbell, and read about his "follow your bliss' mantra and never forgot it. Usually...organization/position exist yet?", you are in the early stages of creating an opportunity. That's how Addgene ...

...remove the tag post-purification. A sample of pCri constructs: pCri-1a, -4a and -9a. 6xHis tags enable affinity...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 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...

...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...interest (POI), that facilitate immunohistochemistry (IHC) experiments with tag-specific antibodies. The most...Viswanathan, 2015). The principal advantage of epitope tags for IHC is the availability of reliable primary... are unavailable entirely. The other advantage is that these tags are small (8-12 amino acids) and neither...

...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...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...backbones for generating other SNAPf-, CLIPf-, and CLAP-tagged proteins for mammalian cell expression. These plasmids...

...Roux lab has deposited HA tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions...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 ...expression screen that can be used to generate His-tagged human kinase constructs that express well in ...blotting and immunohistochemistry, and are summarized in the table below:   ID Cv (Capsid) FLAG Tag Terminus...technology. To streamline CRISPR/Cas9-based genetic tag insertion in the C. elegans genome, Erik Jorgensen...119 selectable marker, and “connectors” that link tags to the targeted gene are supplied to the reaction...

...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...expression patterns. The lab has also used CellTags for tagging two cell populations in a competitive transplant....    Find CellTag libraries here!  Applications of CellTag The Morris Lab developed CellTag to track the...

... the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis is fairly...  Site-directed mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers (green...Laursen from Cornell University. Site directed mutagenesis is a highly versatile technique that can be used...analysis of proteins (to perform alanine scanning mutagenesis or targeted substitution of key residues), and...relevant in this age of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate...plasmid contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG...Cas9 induced cleavage. Summary of Site Directed Mutagenesis In brief, point-mutations can be introduced to...

...supplement of this paper), help each person organize his or her thoughts to facilitate honest conversation...technique are beneficial for researchers at all stages. Rather than succumbing to academic inertia, it...

...not just to share your riches but to reveal to him his own." -Benjamin Disraeli Mentor, Sponsor, Advisor...being coddling, to listen without being taken advantage of, and to respect my students as developing colleagues...

...guest blogger Robert Orr! Robert Orr recently earned his Ph.D. in Biology and Biotechnology from Worcester...polarized cell growth in the lab of Luis Vidali. For his next adventure, he will join the lab of Darren Gilmour...tool requires careful balancing of the inherent advantages and limitations of any technique in the context...technologies, one might incorrectly relegate RNAi to history. The straightforwardness of RNAi is unmatched—unlike...cannot be used to investigate essential genes. Historically, gene silencing methods were thought to be afflicted...interest. Tools for RNAi Arguably the greatest advantage of RNAi is the straightforward nature of the technique—all...approaches and considerations present at the first stage of an RNAi-based investigation. Creation of the ...

... 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...a nucleus -> DNA compaction around histones -> Examples of histone-modifying enzymes and reader-effector...

...under a lot of pressure recently which had affected his judgement. Eventually, we published the paper in ... your structure. Develop your protagonist In most cases, the protagonist will be you. Make sure to highlight...enjoy, remember, and listen to narratives — take advantage of that in your work The part of my job I most...

...Communication where he completed a master's degree in 2020, his research explores risk-taking behavior among collision...into a summer school course at the University of Otago’s Department of Science Communication the following... The Owheo building, home of the University of Otago's Science Communication Department The power ...Alex Gilbert is a PhD student at the University of Otago. Studying in the same Department of Science Communication...

...development, teaching, etc. Church also encourages his mentees to follow their passion. “There are many ...interviewed Addgene depositors who are at various stages in their careers to get advice on choosing a lab...

...epitope tag (6xHis or GST) to affinity purify the expressed protein with a specific resin (e.g. 6xHis containing...protein available – you can always add an epitope tag or a fusion protein. Mammalian vectors One of our...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 ...level expression? Finally, you can add bells and whistles to your construct. If you are planning to track...be a more robust expression system and has its advantages; however, remember that you will need accessory...containing pDEST-HisMBP). After purification the protein can be released from the resin and used for various...

... Several adaptations of this method have been to tag, cage, uncage, and trap the chemical fluor with the...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...earliest developed voltage sensor with a genetic component. The VSD components have historically come from a ...occurring voltage indicator. To give an example, a genetically encoded phosphatase (voltage sensing) hydrolyzes...form of fluorescence or voltage indicators.   Viral Vectors and Voltage Indicators As previously...previously mentioned, cellular voltage is measured at the cell membrane, and this voltage is particularly physiologically...

... 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. ...separately. The proteins, which are purified via histidine tags, are optimized for SDS-PAGE gels and can be...b) Schematic of anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of...blotting, and more. Given the popularity of FLAG and HA tags, you may not even need to clone new constructs to...editing within a single expression cassette is advantageous to Cas9’s larger expression requirements (Figure...