...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...contains an HA-binding scFv that’s fused to a fluorescent protein such as GFP. The HA frankenbody works just...which uses an scFv to amplify the fluorescence intensity of a tagged protein. Resources on Addgene.org: ...often genetically fused to other proteins, which has led to their application for basic and translational ... research applications of scFvs: The SunTag system uses an scFv to amplify the fluorescence intensity... are often the building blocks for engineering proteins. So let’s get acquainted with scFvs. What is an...antibody.  The ability to combine scFvs with other protein domains, much like lego pieces, makes them better...

...C terminus of a protein may disrupt the biological function of some proteins. Protein folding issues, ...of detection of a mutant protein vs. endogenous WT levels of the same protein when both are expressed ...to a protein’s termini to increase detection. The tag can also be removed by treating the protein with...validate tagged protein functionality (Arribere, et al). In rare cases, tags can also affect protein localization...of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol...antibody that works for your application, specifically detects your protein, is species compatible, and...other proteins, quality antibodies do not exist, or they don’t exist for a specific application, or they...

...colleagues identified a highly bright and stable fluorescent protein, StayGold. Although it quickly saw wide interest... E., et al. (2023). A monomeric StayGold fluorescent protein. Nature Biotechnology, 1–4. https://doi.org... al. (2023). Bright and stable monomeric fluorescent proteins derived from StayGold. ResearchSquare (Preprint...recombinant protein, the FLAG tag has proven itself a good option for essentially all applications. Addgene...Lab addressed this problem by optimizing the Cas protein delivery with Peptide-Assisted Genome Editing (...dimerization while maintaining the bright and stable fluorescence of the original (Ivorra-Molla et al., 2023)....mature marmosets. CAP-Mac also robustly expressed fluorescent reporters in green monkeys and cultured human...

...chimeric protein consisting of Cas9 and a photo-dissociable dimeric green fluorescent protein (pdDronpa...CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create...composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1...form a functional protein due to being fused with high-affinity interacting proteins Coh2 and DocS, respectively... saCas9 protein. Stop light! Photoactive anti-CRISPRs halt Cas9 activity Anti-CRISPR proteins are a highly... architecture (Hoffman et al., 2020). Fluorescent dCas9 proteins can be used to identify specific genomic...telomere-targeting gRNA and a dCas9 fusion protein with three red fluorescent tags in U2OS cells. Upon light activation...

...plasmids/viruses are needed to deliver all the protein and RNA components, and several sgRNAs must be ... start sites, all of which yield the same BDNF protein. To demonstrate the specificity that can be achieved...The pooled nature of this experiment requires fluorescence activated cell sorting (FACS) to isolate differentiated...inserted human CD8, a gene that encodes a surface protein for which there are good flow cytometric antibodies...recent advancements and offer commentary on their application to neuroscience research. What is CRISPR-based...accessible to neuroscientists, not all neuroscience applications require such optimized tools. This is elegantly...study neuronal differentiation, it could also be applied to the differentiation of other CNS cell types,...

...cells in live imaging application. Each visual barcode consists of a fluorescent protein coupled to a specific...GFP-nanobody rather than a specific protein of interest. This fusion protein can then be co-expressed in essentially...stabilizes the TurboID-nanobody protein and allows TurboID to start labeling proteins in close proximity to the...localization peptides with four different fluorescent proteins, and inserting each visual barcode into ...reporters that drive the expression of the fluorescent protein by a specific transcription response element...approach has been hampered by the need to tag each protein of interest and, for in vivo work, generate a dedicated...zebrafish line expressing a GFP-tagged version of a protein of interest. The binding of the GFP nanobody to...

...tagging proteins, as it can be fused to the N- or C-terminus (or even in the middle of proteins) and performs...imaging in C. elegans By Alyssa Neuhaus Red fluorescent proteins like mScarlet are essential tools for visualizing...under CC-BY license.  These optimized fluorescent proteins will be a useful addition to the toolkit... mScarlet3 for applications needing intense brightness (such as low-expression proteins of interest) and... produced by our partners at the Institute for Protein Innovation (IPI), part of a new collection of epitope...short, linear epitope derived from the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987)...including popular new approaches like PROTAC targeted protein degraders. But CRBN has been difficult to study...

...decorated with a fluorescent protein remain functional. Attaching the fluorescent protein fusions allowed...produced by orchestrated movements of networks of proteins consuming molecular fuel sources. While the importance... can be generated by conformational changes in proteins? How can information propagate through a structured...rupture E. coli spheroplasts to release encapsulated proteins. Video by ACS. For a more cartoony explanation...addressed using R bodies: simple, force-generating protein machines that can extend and retract over many ...

... decade, but the fluorescent proteins (FPs) necessary for many of these applications have lagged. Fixation...Campbell, B.C., et al. Chemically stable fluorescent proteins for advanced microscopy. Nat Methods 19,...immunoassays due to only having one binding site per protein. Having multiple antibodies allows you to use them...light and electron microscopy and observed high fluorescence resilience to these preparations. After crystalizing...    Fig. 4: hfYFP retains folding and fluorescence in high molarity GdnHCl, at high temperatures...antibodies may just not perform as well in one application as another, so it is always best to have options...for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page for more...

...cleaves the supplied fluorescently labelled ssDNA substrates. Upon cleavage, the fluorescence is no longer quenched... cleavage of fluorescently labelled RNA, the authors employ a P51 cardboard fluorescence visualizer that...binding, changes in fluorescence can be visualised. Another approach was to apply the same concept to ...streptavidin portion of the probe via biotin. The fluorescent portion of the probe that is cut will continue...and released when cleaved. Cleavage results in fluorescence and this approach can be incorporated into a...test varies in the sequence of steps and the Cas protein used. Here, all the components needed for the reaction...recombinase, and the ssDNA-FQ reporters that exhibit fluorescence upon cleavage. Upon amplification by the RPA...

...isolate a protein, there must be a special feature about that protein that no other protein in the expression...(FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry, etc.). Fluorescent tags are primarily used...affinity tags can make protein purification a cinch. Types of affinity tags, the proteins they work best attached...common tags available. Maltose binding protein Maltose binding protein (MBP) is a 45 kDa tag which can be ...unlikely to perturb protein function. This makes it amenable to being left on your protein of interest, especially...you want the tag to remain on your protein for downstream applications Below, we describe a few of the ...purification, so as not to perturb protein function for downstream applications. Tag cleavage must be performed...

...AID-tagged protein. To quantify protein degradation the Ward lab used fluorescent proteins (FPs) fused...implications for the fusion protein effectivity), and a fluorescent protein tag for visualization (Fig....catalytically inactive dCas9) to modify chromatin. A fluorescent protein can still be included for visualization purposes...attached to your protein of interest. TIR1 interacts with endogenous Skp1 and Cul3 proteins to form a SCF...inducible degron sequence) tagged proteins. Degradation of your protein interest would also result in depletion... histone-modifying enzymes and reader-effector proteins. b) Golden Gate assembly (backbones available ... create multiple synthetic chromatin-modifying proteins quickly and accurately.   Priode and Haynes, ...

...in a direct or unbiased way at the DNA, RNA, or protein level. The “correct” choice of tool requires careful...unmatched—unlike CRISPR experiments that require exogenous protein, successful induction of RNAi only requires the...and/or fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples...Non-destructive methods, most popularly with a fluorescent reporter, directly couple silencing of your gene... that contains a target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore...have readily observable phenotypes quantified. Fluorescent reporter-based RNAi constructs have been designed...presence of the RNAi transgene, followed by a fluorescence readout of RNAi activity. As a plant researcher...

...signs of toxicity or burden relative to a fluorescent protein control. We also added the toxin-antitoxin...major structural protein GvpA, which seemed overexpressed compared to accessory proteins. Excess gvpA plasmid...What are Acoustic Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used...of ARGs results in the production of air-filled protein nanostructures called gas vesicles, which scatter.... coli. We deleted an unannotated hypothetical protein, improving expression, and named this gene set ...in mammalian cells without the shell stiffening protein GvpC, which allowed the GVs to be flexible and ...ultrasound imaging of nanoscale acoustic biomolecules. Appl. Phys. Lett. 110, 073704 (2017). Hurt, R. C. et ...

...locations. C) Enrichment of dual-labeled proteins identifies proteins that had been trafficked from the “source...generated a fusion protein called iPAK4 that assembles into a slow-growing protein fiber in the cytoplasm... Figure 4: Time-resolved protein ticker tape. A) Design of iPAK4 protein ticker tape recording. B) HEK...labeling can identify protein interactions or map a local proteome, but some applications have been challenging...labeling and TransitID Tracking cellular history with protein ticker tape Teaching an old antibody new tricks...recently used phage-assisted continuous evolution and protein engineering to generate seven new prime editors...., et al. (2023). Phage-assisted evolution and protein engineering yield compact, efficient prime editors...

...SL-sensitive fluorophore (circularly permuted green fluorescent protein or cpGFP) to the petunia SL enzymatic receptor...relies on the fluorescence ratio between the 2 fluorophores, rather than on the fluorescence intensity, ...thoughtfully designed plasmid components with Fluorescence Activated Cell Sorting (FACS), allowing for .../doi.org/10.1101/2020.05.04.076067    Novel fluorescent enzyme biosensors provide direct, specific and... Claudia Vickers’s lab has designed novel SL fluorescent biosensors by integrating a SL-insensitive fluorophore...conformational change of the receptor which modulates the fluorescence intensity of the cpGFP without altering that... authors also showed that the receptor-based fluorescent biosensor has a high sensitivity (50 nM-500nM...

...containing two fluorescent proteins and four repeats of a miRNA target site. One fluorescent protein (EBFP2) ...B (BoNT/B), which cleaves the synaptic protein VAMP2, a protein essential for synaptic transmission. Cleavage...and function of microbes rely heavily on fluorescent protein genes. These optical reporters have limited...gas-filled protein structure that water-bound photosynthetic organisms use for buoyancy. These proteins allow...established pair of proteins (iLID and SspB) that undergo light-induced dimerization. Upon application of blue light... lab discovered in 2014 that these gas vesicle proteins scatter sound waves and create ultrasound contrast...

...photostable than fluorescent proteins, making them ideal for single-molecule fluorescence techniques. After...CLIP-SNAP fusion proteins for single-molecule FRET. A) Schematic of a CLIP-SNAP fusion protein (a.k.a. CLAP... a flexible linker where a protein of interest can be inserted. B) Protein expression and labeling procedure...optimized strategy uses new combinations of PE protein components (Figure 1), an altered epegRNA delivered...study the conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of...FRET to study the intramolecular dynamics of the proteins in solution and when associated with microtubules...generating other SNAPf-, CLIPf-, and CLAP-tagged proteins for mammalian cell expression. These plasmids ...

...and neuron-specific transgene expression. Fluorescent proteins are widely used for tracing and their expression...2017) that express one of a few different fluorescent proteins (red, green, blue). If a researcher injects...Maday et al., 2014). It is currently unclear which proteins facilitate transport of neuronal tracing compounds...either produce a colorimetric product or are fluorescent. While dyes usually spread unidirectionally, ...biotinylated dextran amines (Veenman et al., 1992) or fluorescent molecules like rhodamine-isothiocyanate (RITC... For predominantly retrograde tracing, albumin protein labelled with horseradish peroxidase (HRP; Kristensson...germ agglutinin WGA, Schwab et al., 1978) or fluorescent molecules such as Fluoro-Gold (Schmued and Fallon...

...Burry 2011). Autofluorescence Endogenous fluorescence of the sample, or autofluorescence, also interferes...example, if you are studying the effect of a new tau protein kinase inhibitor on slowing diseases progression...experimental (?) groups were run on a western against a protein standard (P) to check for tau molecular weight ...experimental protocol but does not receive either protein kinase inhibitor. Since the negative control group...positively charged groups with generally sticky proteins such as bovine serum albumin. Secondary antibodies...than the background noise (Waters 2009). If the fluorescence from your target is extremely bright, then the... the noise likely will not interfere. If the fluorescence from your target is weak, then it is best to...

...Control Protein Activity Learn more about protein tags Read about E. coli strains for protein expression...expressed in bacteria and extracted with common protein purification methods. Read on to learn more about... distance between the target antigen and the fluorescent label, which can reduce the potential image resolution... resolution of super-resolution fluorescence microscopy. Their size also limits their ability to penetrate...be better suited for the job. As single domain proteins, they can be expressed in bacteria, making nanobodies...penetration and decreases the distance between a fluorescent tag and the target antigen to about 2 nm, which...: Purified nanobodies are then tagged with a fluorescent dye or HRP via maleimide-labeling. Maleimide-...

...quenched fluorescent ssRNA reporter is added to the reaction. Cleavage of the quenchable fluorescent RNA by...few years, scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple,...has adapted natural RNase activity of the Cas13 protein to develop and optimize the method termed Specific...Cas13 and Cas12a, are combined with different fluorescent reporters for each enzyme. In addition, Csm6,...Csm6 and a Csm6 specific reporter (in the same fluorescent channel as the Cas13 reporter), they could significantly...papilloma virus (HPV) genome, via a crRNA. An ssDNA-fluorescently quenched (FQ) reporter, which will produce a...rate of ~1,250 cuts per second. A quantifiable fluorescent signal designates the presence of your DNA of...

...a programmable RNA-binding protein, creating the fluorescent fusion protein dLwaCas13a-NF for in vivo ...has many applications. That said, SpCas9 is not the only game in town, and other Cas proteins like SaCas9...form of “bait” to identify new CRISPR-associated proteins within the bacterial genome. From this analysis...categories based on the architecture of the CRISPR protein in question: C2c1, C2c2 and C2c3 (short for Class...consistent with known roles for HEPN domains in other proteins. As with Cas9, mutating key residues in the Cas13a... the sequence of interest and an inactivated fluorescent RNA reporter. If the target sequence is present...resulting in activation of the fluorophore. The fluorescent signal can be used as an indicator to determine...

...Developments in light sheet fluorescence microscopy Modern light sheet fluorescence microscopy was first pioneered...such as whole mouse brains expressing green fluorescent protein (GFP) (6).   The Dodt and Frank Bradke laboratories... advances in microscopy has been light sheet fluorescence microscopy (LSFM). The underlying method was...colleagues and originally named orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David...laser beam into a thin sheet to illuminate a fluorescent sample and captured the reflected light using...configurations’ various features see (4,7).   Light sheet fluorescence microscopy vs. confocal microscopy In confocal...developed AAV (adeno-associated virus)-based fluorescent labeling methods that can be used with 3DISCO...

...integration of fluorescent tags in neurons. Their toolkit named ORANGE (Open Resource for the Application of Neuronal...engineered SECURE adenine base editor variants using a protein-truncation strategy with reduced RNA-recognition...abilities of C-to-T and A-to-G base editors into one protein. They fused an activation-induced cytidine deaminase...Editing) mediates targeted genomic integration of fluorescent tags in neurons.  Read the paper in bioRxiv ...with a com aptamer which binds an aptamer binding protein for packaging into a lentivirus-like particle. ...tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase for RNA editing...bioRxiv Find the ORANGE toolkit   Cas9/gRNA ribonucleoprotein delivery by lentiviral capsid-based bionanoparticle...

...then purified from the supernatant using Protein A or Protein G affinity columns. Formulation Buffer After...measured and the ratio of the antibody protein content to the total protein content of the sample is determined...and a species-specific fluorescently-labeled secondary antibody. The fluorescence pattern is compared to...secondary antibody. Protein expression is compared to an untransfected control and the protein size is confirmed...by Addgene were developed by the Institute for Protein Innovation (IPI). For these antibodies, IPI will...these antibodies, please visit the Institute for Protein Innovation Antibody Collection webpage. Antibody...concentration and stained with Coomassie blue. Protein band intensities are measured using ImageJ software...

...fused to a fluorescent protein (FP) can help visualize specific genomic loci using fluorescent microscopy...Catalytically dead dCas9 fused to a cytidine deaminase protein becomes a specific base editor that can alter DNA...purify a region of genomic DNA and its associated proteins, RNA, and DNA. The enCHIP system uses an anti-... avoiding known transcription factor and other protein binding sites. ID Plasmid Gene/Insert Promoter ...may also be fused to gRNA sequences to produce fluorescence at a target region, as in the CRISPRainbow kit...using CRISPR, you will need to express both a Cas protein and a target-specific gRNA in the same cell at ...Single plasmids containing both the gRNA and Cas protein act as all-in-one vectors, but their function is...

... DNA repair protein, and is suitable for both cell-permeant and non-cell-permeant proteins. CLIP-tag CLIP-tag...complementary protein function or localization. ACP and MCP tags ACP- and MCP-tags are small protein tags based... labeling cell-surface proteins, and are useful for labeling secreted proteins with disulfide bridges ... Users may still generate their own fluorescent or non-fluorescent CoA substrates by purchasing coenzyme...internal or surface proteins Single constructs are compatible with multiple applications (different fluorophores...ACP or MCP tagged plasmids for visualization of proteins inside cells. Depositor...Information New England Biolabs offers non-toxic protein labeling systems based on SNAP-, CLIP-, ACP-, and...

...mini counterpart label nearby proteins with biotin. The biotinylated proteins can then be isolated and identified...for Glypicans DNA editing with STITCHR Dynamic protein labeling with TurboCas Enhancer-AAV viral preps...transporters. The intensity-based Glutamate-Sensing Fluorescent Reporter (iGluSnFR) designed and improved by ...primary visual cortex (V1) layer 2/3 (L2/3). B) Fluorescence from axonal boutons of neurons expressing iGluSnFR3...our bacterial plasmid set for expressing tagged proteins, and stay tuned for mammalian expression and AAV...therapeutic potential. Our partners at the Institute for Protein Innovation (IPI) recently released a set of highly-specific...antibodies against three of the six human glypican proteins (GPC1, GPC3, and GPC4). All IPI antibodies undergo...

...to change protein levels? Until recently, no such tool existed for rapid, customizable protein degradation...both short-and long-lived proteins, and it can even distinguish between protein variants when used with ...addition of exogenous TRIM21 protein along with an antibody targeting the protein of interest led to rapid...has deposited plasmids encoding fluorescent reporters and other proteins controlled by GAL4 and ZIF268....James and Schuh labs. Clift et al. applied TRIM-away to nine proteins in ten ddifferent cell types, including...Plasmids Podcast! TRIM-Away: Targeted endogenous protein degradation Article contributed by Mary Gearing... cell populations, co-microinjection of TRIM21 protein and the target antibody works well. In larger cell...