...graphics. PS: they’re free. 3. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection...Monomeric Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi by Jennifer Tsang Learn about mNeonGreen, mTFP1..., optogenetics, chemogenetics, plasmids, fluorescent proteins, careers, and more. These resources can ...Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins by Cary Valley, Shreya...mWasabi and find plasmids containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk...strategies for generating GFP fusion proteins. From considering protein size and shape, linker sequences,...chalk talk, and tips for practicing.  6. The Fluorescent Vegetables in Aptamer Soup by Aliyah Weinstein...

... of a fusion protein: Histidine tag, malate dehydrogenase, and green fluorescent protein. Biochemistry... graduate school. As a stable, easily purified protein with easily measured activity, MDH is particularly...structure, function, and interaction with other proteins.   It is important to note that research ...

... blog Fluorescent Proteins 101: Fluorescent Biosensors Resources on Addgene.org Fluorescent protein...short), where a fluorescent protein is inserted into one of the loop regions of any G-protein-coupled receptor...fusing two bright fluorescent proteins with different thermal quenching rates (mNeonGreen and tdTomato), ...NIR calcium reporter Near-infrared (NIR) fluorescent proteins promise better performance for in vivo use...calcium indicator using small monomeric NIR fluorescent proteins (miRFP670nano and miRFP720) and the Ca2+...engineering of potassium binding proteins, they developed a family of fluorescent potassium indicators with ...protein plasmids and resources Fluorescent protein guide: Biosensors Collection ...

...'ll find in this post: Yeast-based protein evolution Fluorescent tools for mammalian cells Nanobody purification...enables tracking of protease activity with a fluorescent protein--no affinity purification required! The team...cells By Angela Abitua Genetically encoded fluorescent proteins are often used to label  specific structures...biomolecules within cells. However, sometimes these fluorescent proteins might not localize to the right places, ...example, to improve nuclear localization of fluorescent proteins, they found that adding a triple nuclear...Find the sensors at Addgene! New yellow fluorescence protein-based calcium sensors (jYCaMP1), variants...nm and enable two-color imaging with red fluorescence protein-based calcium sensors. These new calcium...

...photoswitchable elements into many other types of proteins. Here, we’ll highlight a few recently-deposited...license. A naturally occurring microtubule-severing protein, Katanin is of course named for the samurai sword...optogenetic module to activate or recruit your protein of interest, maybe you want to inhibit it. Restricting...development and a knockout is not feasible or when the protein of interest supports a variety of processes in ...CIB1 clustering. Fuse either CRY2 or CIB1 to your protein of interest and express the other separately, then...generate CRY2-CIB1 oligomers that trap the fusion protein away from its usual location, preventing it from...such a strategy to dissect the roles of three Rab proteins during a key stage of zebrafish development (Aljiboury...

...researchers from some labs have developed fluorescent-protein based DA sensors (Figure 2). The dLight1 ...sensors, the new dLight1 sensor is a single fluorescent protein-based sensor. This sensor was engineered ...a GFP variant and a DA receptor such that GFP fluoresces when the receptor is bound by DA. Two variants...affinity and high-magnitude responses (increases in fluorescence) at low concentrations (e.g., 100 nm) of DA....variant bound to a DA receptor, such that the GFP fluoresces upon DA-receptor binding (Sun et al., 2018). ...These sensors also exhibit high magnitudes of fluorescent responses. Studies on the kinetics of these sensors...Sun, Fangmiao, et al. "A genetically encoded fluorescent sensor enables rapid and specific detection of...

...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...

...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 ...

...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...application. Each visual barcode consists of a fluorescent protein coupled to a specific subcellular localization...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...

...of indels and thus frameshifts that disrupt the protein-coding capacity of a locus. When using S. pyogenes...code for amino acids near the N’ terminus of the protein, in order to mitigate the ability of the cell to...for amino acids close to the C’ terminus of the protein, to maximize the chances of creating a non-functional...nucleotides, restricting gRNAs to 5 – 65% of the protein coding region will still result in many dozens ... a specific edit, such as the insertion of a fluorescent tag or the introduction of a specific mutation...0561-9 Dempster JM, Pacini C, Pantel S, Behan FM, Green T, Krill-Burger J, Beaver CM, Younger ST, Zhivich...

... be used as a proxy measure for differences in protein expression. (If you're just getting started, check...fluorogenic primers Low Single self-quenching fluorescent molecule connected to a primer  No Yes ...normalize for that. Passive reference dyes are inert fluorescent dyes that are not dependent on amplification ... It is possible to multiplex qPCR when using fluorescent probe-based chemistry - theoretically, up to ... Tip🔥 The ABI machines automatically record fluorescent data on all channels - helpful if you forget ...quantification of relative mRNA abundances using SYBR Green I and real-time RT-PCR. J. Immunol. Methods 283:...

...method for identifying protein-protein associations. This method uses a bait protein fused to a biotin ligase...PrM N and C   Detecting protein-protein interactions with DULIP Proteins rarely act alone. Molecular...which “bait” proteins are tagged with Protein A-Renilla luciferase and the “prey” proteins are tagged with...with Rosella, a pH-sensitive fluorescent biosensor A pH-sensitive fluorescent biosensor-based assay system...ligase to biotinylate proximate proteins in the cell. These candidate proteins can be enriched via biotin ...of fusion proteins, requires less biotin, and shows enhanced labeling of proximate proteins. Overall BioID2...BioID2 improves the efficiency of screening for protein–protein interactions. The Roux lab has deposited HA...

...sequences for several different fluorescent proteins. Visualizing fluorescence can be challenging in plants...because of the auto-fluorescence of chlorophyll, so we looked at newer fluorescent proteins that get around...construct on the same piece of DNA. We do a lot of protein-protein interaction work and we’ve gotten some tools...Reading: Quick, Versatile Plant Transgenesis with GreenGate Plasmids  ...

...molecule, the antibody binds to a protein, and then voila! Wherever your protein of interest is, a signal will...signaling molecules are represented in light green; and proteins are represented in orange. Created with BioRender.com...molecules to identify each protein of interest, this gel-based method identifies proteins solely by weight and... isn’t giving you enough signal to detect your protein of interest, it may be time to look at the indirect...antibodies. While a primary antibody binds to the protein of interest, a secondary antibody binds to the ...signaling molecules associated, indirectly, with each protein of interest.    Figure 1: The direct ...three, secondary antibodies are indicated when the protein of interest is found in low amounts or if the conjugated...

...absence of a protein, determine if a protein is up or downregulated, examine a protein’s stability or ...Hjelm, 1972, Bjorck 1984). Protein A, Protein G, or the recombinant Protein A/G are conjugated to agarose...support while unbound proteins are washed away.  While both Protein A and Protein G have an affinity for...compatible with either Protein A or G.  While commonly used, Protein A and Protein G are not suitable for...applications, such as protein isolation from serum. In this case, Protein A and Protein G will bind both the...of the target protein or interfere with protein interactions of interest. Tagged proteins are typically...modifications, or study how a target protein interacts with other proteins or nucleic acids. Read on to learn...

...well as the type of transgene delivered: fluorescent proteins, optogenetic sensors, CRISPR elements, etc...BBB via interactions with a similar protein called LY6C1. This protein is expressed in both BALB/cJ mice...exists to prevent substances in the blood, like proteins and viruses, from crossing into the brain. While... it, cross the BBB through an endothelial cell protein called LY6A (Hordeaux et al., 2019; Huang et al...capsids that rely on interactions with those two proteins to cross the BBB are often not effective in NHPs...2023 AAV.CAP-Mac CNS: neurons (rats, infant green monkey, rhesus macaque); vasculature (adult marmosets...astrocytes (adult rhesus macaque) Rats, marmosets, green monkeys, rhesus macaques N/A Chuapoco et al....

...expression plasmid involved in coexpression of light (green) and heavy (blue) chains as driven by two CMV promoters... moved to direct functional validation by immunofluorescence-immunocytochemistry (IF-ICC.) This method...exciting for scientists working to visualize multiprotein complexes.  Fig. 2. Multiplex immunolabeling...

... BirA, has been used to identify and study protein-protein interactions in proximity labeling techniques...extensive research history and characterization. Many proteins and processes naturally found in E. coli have ...and a tool-kit from the Hamdoun Lab to create fluorescent fusions in a multitude of organisms including...you are working with Arabidopsis check out the GreenGate Toolkit which allows for easy cloning of constructs...