...rest of the protein in place including FokI endonuclease domain as well as fluorescent proteins used to sort...with the same genetic background and do an apples-to-apples comparison of all those mutations. If you ...mammalian or other cells. TALENs are engineered proteins that comprise a DNA binding domain and an endonuclease...technology allows you to create a pair of designer proteins that will introduce a double-strand break in the...transfection or electroporation. All you do to create a protein that binds those 15 base pairs is take four plasmids...different genetic backgrounds. It becomes more of an apples-to-oranges comparison. Addgene: Can you compare...

...version at Addgene! Fluorescent protein and control AAV These fluorescent protein expression vectors can...exhibits high contrast between peak fluorescence and resting fluorescence. It is useful for activity imaging...exhibits high contrast between peak fluorescence and resting fluorescence.  It is useful for activity imaging...pGP-AAV-syn-FLEX-jGCaMP7b-WPRE) exhibits the brightest resting fluorescence of the GCaMP7 family of calcium sensors and ...the endoplasmic reticulum, but exhibits larger fluorescent responses to GABA. Find the iGABASnFR and its...the control vectors for experiments encoding a fluorescent-tagged molecular tool.   Find pAAV-FLEX-tdTomato...of densely labeled neurons because background fluorescence from inactive neurons is reduced. 105322 ...

...vivo imaging with fluorescent proteins Browse luciferase plasmids Find fluorescent protein biosensors ...tools The luciferase reporter gene The green fluorescent protein reporter gene Resources on Addgene.org ... reporters Limited Depth of Signal/Imaging: Fluorescent reporters are not as well suited for imaging ...context/spatial localization: In vivo imaging with fluorescent or bioluminescent reporters usually only detects...ARGs) are engineered gene clusters that encode proteins that form bacterial gas vesicles. These ~200nm...200nm long vesicles have hollow interiors and a protein shell that excludes water but is gas permeable. These... experiment to demonstrate the most exciting application for ARG’s: in vivo imaging. Specifically, arg1...

...can visualize the protein via fluorescence or chemiluminescence, precipitate the protein out of solution... a single protein that can recognize the target protein (Wang et al., 2013). These proteins are also plasmid-derived...to study protein-protein interactions. Flow cytometry Flow cytometry is used to detect proteins on the ...scientists to target specific proteins they’re interested in. Once a protein is targeted with an antibody...entirety of the target protein: instead, it recognizes a small portion of the protein, known as an epitope... the target protein because the antibodies will recognize multiple epitopes on the protein. However, each...with other, similar proteins due to recognizing a single epitope of the target protein. However, small mutations...

...Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization...Research Campus reminds us that, although fluorescent proteins are incredibly useful, chemical fluorophores...different pH’s and some that are fused to fluorescent proteins. She also showed that R-bodies can rupture...Zika virus and, upon binding, produce reporter proteins. These toehold switches can be used to quickly...University, describes R-bodies, “force generating protein machines that can extend and retract over many ...Casilio, a system that uses modular RNA binding proteins, dCas9, and a modified gRNA to direct a variety...genes in a single cell, and localization of a fluorescent signal to your target gene. Read this post to...

...for the amount of protein you are expecting.  Fluorescent proteins Fluorescent proteins, while more expensive...binding to your protein of interest. Protein preparation Before you can blot your proteins, you’ll need ...normalize your protein of interest to overall protein expression for each sample and protein. While ubiquitously...normalize your protein of interest to total protein expression. Total protein expression can be measured using...rely on primary antibodies conjugated to fluorescent proteins, instead of the more common chemiluminescent...consider the subcellular location of your protein. Proteins located in membrane-bound subcompartments,...considered if your protein is difficult to solubilize, such as a membrane-bound protein. There are many ...

...Indirect labeling is achieved through protein-tag or protein-protein interactions, like biotin-streptavidin...transmembrane proteins, such as receptors, or post-translational modifications on those proteins, like glycosylation...kits allow for labeling of cytosolic proteins, as well as proteins of the secretory pathway. Those can ...buffer to label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear...that bind DNA or free amines of proteins. Depending on the application, they can be membrane-permeant ...like many of the cluster of differentiation (CD) proteins found on immune cells. However, there are more...methods can include labeling markers with multiple proteins/antibodies, targeting markers found within the...

...antigens: GFP, RFP,  the histone protein H2A2B, and the actin-binding protein gelsolin, but any nanobody sequence...tissue sections Amplifying weak signals of fluorescent protein (i.e. GFP, RFP) Detecting GFP reconstitution...Antibodies are a go-to tool for detecting a protein of interest in cells and tissues. Although antibody...and instead are conjugated to reporters (i.e. fluorescent dyes, HRP, etc. ) after purification. Antibodies...signal amplification occurs when HRP converts a fluorescently labeled tyramide molecule into a highly reactive...linked to residues within close proximity thus fluorescently labeling them. HRP RANbody staining results ... ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to lower background...

...structured surfaces. Plasmids expressing fluorescent proteins will be used to tag individual strains of...condition is crucial. Plasmids expressing fluorescent proteins and a new two-photon laser scanning microscope...containing promoter regions that activate fluorescent protein transcription in response to river contaminants...within the biofilms will then be monitored using fluorescence microscopy and FACS. In an additional approach...complications of diabetes mellitus. Recombinant human proteins that play a role in insulin regulation and diabetes...and the activity of the plant compounds on these proteins will be tested. Investigating changes in neuronal...water samples at specific locations, measure fluorescent signals from the sensor, and send a signal including...

..., chemical dyes, and genetically encoded fluorescent proteins (Gambarotto, et al. 2019; Tillberg, et al...Stiles MA (2022) Improved fluorescent signal in expansion microscopy using fluorescent Fab fragment secondary...2016) Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins...workings of the synapse by conventional light or fluorescence microscopy (Dani, et al., 2010). How do you ...the resolution barrier while still utilizing fluorescence microscopy, is another option. However, most...samples. Reduced signal can be caused by loss of protein during digestion, or masking of epitopes during...adapted for detecting nucleic acids as well as proteins (Klimas, et al. 2023; Wassie, et al. 2019). Perhaps...

...mCherry fluorescent protein followed by a stop codon and the coding sequence for a green fluorescent protein...of cell-free system, capable of producing fluorescent proteins and enzymes. Image from Moore et al., 2021...validated through monitoring the expression of fluorescent proteins and oxytetracycline enzymes. Next, this ...biochemical secondary energy sources to increase protein yields from 36 μg/mL to 266 μg/mL. The reaction...libraries: Cas13X and Cas13Y, two compact Cas13 proteins found in the metagenome datasets from hypersaline...messenger-RBP sgRNA Library targets human mRNA binding proteins with 10 guide RNAs per gene. The lab modified ...several MYC-dependent cancer cell lines. It can be applied for identification of essential MYC binding sites...

... et al. "Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization...the proteins they label. In fact, a recent study of 400 proteins found that 20% of tagged proteins did... they compromised protein-protein interactions. If you’re looking to a tag a protein, inntags IT5 and ...examining protein-protein interactions, particularly in single-molecule or high-throughput applications. Perhaps...tag may interfere with protein localization and/or function, so each tagged protein must be tested carefully... other Protein Tags in our Plasmids 101 Series. Get the Inntag Plasmids Tag endogenous proteins with CRISPR... CRISPR Fluorescently Tag Your Protein of Interest ...

...bacteria CRISPR-Casɸ mGold, a new yellow fluorescent protein New CRISPR plasmids New items from the viral...32675376/ mGold, the most photostable yellow fluorescent protein to date By Justin Ng High-throughput screening...cell types expressing variants of Yellow Fluorescent Protein (YFP). The team identified a new YFP variant...splitting and joining the protein regions with Vivid (Vvd) photodimers. Upon the application of blue light, Vvd...use light to induce conformational changes in a protein.    Cells expressing an RFP reporter plasmid...screening methods, such as fluorescence-activated cell sorting (FACS) helps researchers sift through thousands...Resources page.  CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair) is a double-strand break repair assay...

...recent contribution to the field is a series of fluorescence resonance energy transfer (FRET)-based calcium...colleagues began by isolating a calcium-binding protein, troponin C (TnC), from muscle cells. They identified...calcium binding and developed a version of the protein with as few calcium-binding sites as possible for...transcription factor cAMP-responsive element-binding protein (CREB) in living cells. “Now we are making this...Eindhoven has similar interest in FRET-based sensor proteins, particularly for the intracellular imaging of...with a solution: they devised self-associating fluorescent domains whose association is disrupted in the...Robust red FRET sensors using self-associating fluorescent domains. ACS Chem Biol. 2013 Oct 18;8(10):2133...

...the core of the protein. OptoNBs were also tagged with an infrared fluorescent protein tag which let the...choose your own protein purification adventure. You want to purify your favorite protein (YFP). You have...bind and release a protein of interest in response to light allows for tagless protein purification as well...purification of unmodified proteins. Affinity purification methods typically require a protein to be tagged, which...approaches to use with recombinant proteins. Purifying an untagged protein with a flip of a light switch would...to light-controlled protein binding, they probably don’t bind your favorite protein yet. More OptoNBs would...binding.  Light controlled protein purification with OptoNBs The first application for OptoNBs takes us in...

...DNA Fluorescence dyes Another way to quantify DNA would be to use fluorescent dyes that fluoresce when...and their corresponding fluorescence. That way, you can compare the fluorescence of your sample against...acids (DNA and RNA) absorb maximally at 260 nm. Proteins on the other hand absorb best at 280 nm and organic...sequencing. Unlike absorbance-based methods, fluorescence-based methods require a standard curve, a set...this method doesn’t tell you about contaminating proteins or chaotropic salts. Capillary electrophoresis...These fragments are quantified over time using a fluorescent dye that intercalates into the DNA such that ... using capillary electrophoresis which shows fluorescence units over time. Smaller fragments migrate more...

...Which Fluorescent Protein Should I Use? Plasmids 101: Green Fluorescent Protein (GFP) Fluorescent Protein...leash for your protein. If you’re working with an uncharacterized protein, or a protein for which a good... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection...fusion protein tags. Biochemists and molecular biologists who need to overexpress and purify proteins can...tags and fusion proteins to aid in the expression and purification of recombinant proteins. Tags for stability...recombinant protein? It is not generally in a cell’s best interest to overexpress a protein. Energy and...

...MitoTimer proteins are derived from the Timer protein - a mutant of the fluorescent protein dsRed that...three possible fluorescent proteins, chosen for minimal spectral overlap, minimal protein aggregation, ...groups to monitor protein and cell ageing, as the spectrum shift occurs during Timer protein life due to a... synthesized (green) protein and organelles with older (yellow to orange) protein. Image courtesy of Roberta...1852-61. A quicker method for protein inactivation Your protein of interest does x, but it also participates...your endogenous protein of interest and co-expressing its replacement recombinant protein with a FKBP domain...outer membrane protein with a FRB domain (MitoTRAP). Rapamycin binds your recombinant protein (via FKBP) ...

...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...