... number of fluorescent proteins is steadily increasing. Often, the choice for a fluorescent protein is...refers to the fluorescence intensity of a fluorescent molecule. A higher brightness increases the detected...explains how to critically assess the reported properties of fluorescent proteins, how to do a head-to-head...comparison of fluorescent proteins and how to make a well-informed decision on the best fluorescent protein ...best fluorescent protein for a specific application can seem like a daunting task. Many fluorescent proteins...properties of fluorescent proteins on websites. To make the best choice, the fluorescent protein properties...equipment. Because the performance of a fluorescent protein strongly depends on both the biological system as...

...that’s independent of the reporter. The only caveat is that HA staining is weaker than reporter staining...tagged and instead are conjugated to reporters (i.e. fluorescent dyes, HRP, etc. ) after purification....detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties: HRP, Spaghetti...staining. Picking the right reporter for your RANBody While all three reporters stain cultured cells and ...Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection Method Uses Pros Cons Antigens...antibody and a ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to...proximity thus fluorescently labeling them. HRP RANbody staining results in a ≥10-fold increase in signal ...

...light which revealed a blue light-dependent shift in mCherry fluorescent to the surface of the bead, suggesting...OptoNB-SOS activated Ras/Erk signaling by using a fluorescent reporter for Erk kinase translocation. These results...protein. OptoNBs were also tagged with an infrared fluorescent protein tag which let the team detect its co-...control protein activity Read about RANbodies, reporter nanobody fusions Learn more about using the secondary...signaling pathway.  As a proof-of-principle, the team created an OptoNB to control the Ras/Erk signaling pathway...

...authors have deposited AAV plasmids for Cre-dependent and -independent versions that are trafficked to the...photostable than fluorescent proteins, making them ideal for single-molecule fluorescence techniques. After...used to measure signal along the dendrite. D) Fluorescence signal as a function of the distance between...pharmacological profiling or interrogation of context-dependent signaling in primary cells. The authors used...types in varying conditions, revealing context-dependent responses and molecular patterns of selectivity...Garcia-Marcos, M. (2024). Direct interrogation of context-dependent GPCR activity with a universal biosensor platform...6E10-A5] in Immunohistochemistry (Human). Addgene Report, doi: https://doi.org/10.57733/addgene.6ncijt.  ...

... are two main classes of reporters: chemiluminescence enzymes and fluorescent proteins. Chemiluminescence...amount of protein you are expecting.  Fluorescent proteins Fluorescent proteins, while more expensive, allow...Nitrocellulose or low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased cost (specialty...without increasing background noise and can reduce the amount of primary antibody required. Reporters There...a wide dynamic range for fluorescent proteins. They do tend to have increased background with higher exposure...Indirect detection Increased time; higher background Chemiluminescence reporter Smaller dynamic ...detection method Increased time; higher background Chemiluminescence reporter Smaller dynamic ...

...break (DSB) mediated homology dependent repair by enhanced green fluorescent protein (EGFP) reconstitution...incubation, you assess the level of fluorescence in your cells under a fluorescence microscope. The higher the ...find your mutant from screening as little as 8-10 pups. The level of fluorescence you saw in the validation...digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR, homologous recombination; SSA, single...containing sgRNA sequences into HEK293T cells. The fluorescence intensity was classified into four groups (4...darker than control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted...your target is inserted, disrupting the EGFP fluorescent signal, for details on this plasmid, see Mashiko...

...mCherry fluorescent protein followed by a stop codon and the coding sequence for a green fluorescent protein...expression of fluorescent proteins and oxytetracycline enzymes. Next, this system was used to recreate natural...representation of cell-free system, capable of producing fluorescent proteins and enzymes. Image from Moore et al....plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE, to identify...designed based on MYC-ChIP-seq data from several MYC-dependent cancer cell lines. It can be applied for identification...different biochemical secondary energy sources to increase protein yields from 36 μg/mL to 266 μg/mL. The...

...or fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of ...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...readily observable phenotypes quantified. Fluorescent reporter-based RNAi constructs have been designed...identified as undergoing RNAi through the fluorescent reporter and manually extracted. This process is ...of using a fluorescent output to monitor RNAi, we engineered a positive selection reporter whereby survival...gene target to silencing of the reporter. This is achieved at the level of the DNA construct, where your ...

...thaw cycles. Depending on the researcher and the type of virus, there have been varied reports of titer losses...should appear fluorescent if they were infected. While there may be some low-level fluorescent protein or...Transduction efficiency depends on viral titer Both of these can be increased by concentrating a viral...1. Viral vectors undergo DNA rearrangements. Depending on the composition of the viral vector and the...virus. Alternatively, if the virus encodes a fluorescent protein, the packaging cells could be examined..."Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption... prior to their transduction has been shown to increase AAV transduction efficiency by approximately 15...

...knowledge of the nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing that...oligonucleotides in fluorescent visualization techniques for decades, most notably for fluorescent in situ hybridization...applications including aptamer based fluorescent biosensors. Aptabeacons Fluorescence resonance energy transfer ...aptamers fluoresce until they bind to the appropriate ligand, but relying on a lack of fluorescence for readout...applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing with veggies) Though the...aptamer that would bind the fluorogen and increase its fluorescence, essentially mimicking GFP.  Though the...highly customizable in terms of how they actually “report” as biosensors. Making aptamers glow The flexibility...

... the fluorescent protein genes, Cre recombination is required before fluorescence can occur. Cre recombinase...). Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are organized...of the fluorescent proteins used in the system. One such system is Brainbow 2.0, which uses Cre to trigger...default, they are not fluorescent. Instead, they incorporate a truncated, non-fluorescent form of YFP as the...is necessary to excise the non-fluorescent YFP and activate fluorescence of the downstream OFP, GFP, or...combination of all of the fluorescent proteins from the injected construct. The fluorescent protein expressed ...developing fish via Cre protein microinjection or crossing of Zebrabow animals to an inducible Cre line. The Schier...

...additions, including our first far-red fluorescent marker, a Flp-dependent channelrhodopsin in the retrograde...help, with over 100 user-submitted viral vector reports. And don't forget to use the link below to keep...Biosensor GENIE Project New serotype pENN.AAV.CMVs.Pl.Cre.rBG AAV5 Recombinase James M. Wilson New serotype...

...such as fluorescent microscopy must be used. To learn more about the most suitable fluorescent microscopy... have a variety of fluorescent light sources. When interrogated with a fluorescent light source, a cell...photons varies from cell to cell depending on the level of fluorescence. A cell expressing high levels ...proteins tag your protein of interest with a fluorescent reporter on the N- or C-terminus of the protein. One...conjugated fluorophores Fluorescent antibodies get around the challenges of fluorescent protein fusions by ...laser and interrogated with various visible and fluorescent light sources that allow users to assess a cell... cells in a population in a procedure termed fluorescence activated cell sorting or FACS (2). By analyzing...

...years — a green fluorescent protein, a red fluorescent protein, a fancy new green fluorescent protein, some...new-and-improved fluorescent protein you want to try, just swap in a new fluorescent ligand and see the...purification tags, another version with that same red fluorescent protein somebody made because they forgot it ...of chemical dyes provide many advantages over fluorescent proteins, opening up new applications.   ... self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from Roger Tsien’s lab ...dihydrofolate reductase (eDHFR) could be labeled with fluorescent derivatives of its substrate trimpethoprim (TMP-tag...collection. CLIP-tag: Also in 2008, Kai Johnsson’s lab reported another AGT-based tag named CLIP-tag, which reacts...

...FAST system, Ye’s lab could edit a tdTomato fluorescent reporter gene in mice using Minicircle DNA vectors...the figure below. Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated that...while transcription of the N-terminus is light-dependent (Yu et al., 2020). To express the N-Cas9, Haifeng...protein BldD to the nucleus. To couple this light-dependent activity to transcription, they fused BldD to ...of Cas9 and a photo-dissociable dimeric green fluorescent protein (pdDronpa) (Zhou et al., 2018). pdDronpa... flanking the DNA-binding cleft of Cas9, the fluorescent domains dimerize and prevent DNA binding in the...chromatin architecture (Hoffman et al., 2020). Fluorescent dCas9 proteins can be used to identify specific...

...conjugated with a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such...dish and incubate it with a reporter conjugated primary antibody. The reporter reacts with a provided substrate...capture reagent may be an antigen or an antibody depending on how the assay is set up. Then they block the...peroxidase (HRP) or alkaline phosphatase (AP). Fluorescent tags may also be used, but for simplicity we ...we will discuss the more common enzyme-based reporter methods. For enzymatic signal detection, users provide... sandwich and competitive. In a direct ELISA a reporter enzyme conjugated antibody binds to antigen coated...antibody first binds to the antigen and then a reporter enzyme conjugated secondary antibody binds to ...

...may be a plasmid that constitutively expresses a reporter protein (e.g., GFP or luciferase) that is either... well of your cells. Regardless, the amount of reporter protein activity correlates to both the amount...successfully and expressing the protein. For example, fluorescence microscopy images resulting from our experiment...was not successful due to the absence of GFP fluorescence from the living, viable cells. This result presents...produces the expected effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid...These controls may or may not be plasmid-based depending on the experimental needs. Using our experiment...Plasmid A is the decreased expression of Gene X. Ergo the positive control(s) should decrease the expression...

... is the permanent fluorescent tag portion of Rosella: it will emit red fluorescence regardless of its ...the fluorescent properties of Rosella.   Table 2: Effect of Localization on Rosella’s fluorescence emission...green fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks ...Devenish, R. J. (2008). Rosella: A fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and...mammalian cells using a single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed...Photoconversion Light Sheet Fluorescence Microscopy Additional Resources on Addgene.org Fluorescent Protein Plasmids...Plasmids and Resources Fluorescent Protein Guide: Biosensors Keima, another fluorescent pH-biosensor for studying...

...good reporter genes There are many great reporter genes available and which one you choose depends on your...the T-DNA region with our gene of interest or a reporter gene so that the transformation and gene expression...different treatments on transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase...histochemical GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate...transformation success. Luciferases are great quantitative reporters and use a substrate to produce bioluminescence...half-lives then GUS and are therefore good temporal reporters to measure promoter activity, for example. Useful...tNOS & pSB166 - pL2_pSB90_tMAS::rLUC-I::pMAS. Fluorescent proteins (FP, e.g. GFP) can be used in many ways...

...neomycin, puromycin, hygromycin) and fluorescent protein reporters (GFP, BFP, RFP, no FP). The vectors ...SfiI restriction sites with different overhangs depending upon whether they are on the 3’ or 5’ end of the...region of the RPL41 gene, to drive the selection/reporter parts of each construct. This new tool kit allows... then degraded by the cell.4.) The transposase creates a double strand break in the DNA and allows the...transposase gains its name from the fact that it was recreated from a defective transposase found in fish. This...transposases. Improvements have been made since then by increasing the consensus between a greater number of extinct...primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB tranposase variants for hyperactivity ...