...fluorescence 100-fold compared to other reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains 11...apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11) to increase...self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP...fluorescent executioner caspase reporters. Several apoptosis reporters have been developed, such as the...transfer (FRET)-based executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase...FlipCherry: Red fluorescence protease reporter To add to the fluorogenic reporter toolkit, the Shu Lab also designed...range and high brightness. This reporter achieves approximately 100-fold increase in fluorescence after cleavage...

...halves of EGFP undergo folding, maturation, and fluorescence. Biosensors: A wide array of GFP-based fluorescent...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a promoter of interest.... This type of transcription reporting was among the earliest uses of GFP. Förster resonance energy transfer...expressing GFP from cells that are not. Developmental/transgenic uses: Because of its stability, GFP can be...Fluorescent Protein Mutations Relative to Wildtype GFP EGFP F64L; S65T EYFP S65G; V68L; S72A; T203Y mYFP...from proteins such as Green Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish...

... have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP nanobodies  stain poorly when tagged with...detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties: HRP, Spaghetti...independent of the reporter. The only caveat is that HA staining is weaker than reporter staining. Picking...Picking the right reporter for your RANBody While all three reporters stain cultured cells and tissue sections...Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection Method Uses Pros Cons Antigens...weak signals of fluorescent protein (i.e. GFP, RFP) Detecting GFP reconstitution across synaptic partners...Monster because they recognize this GFP scaffold itself. Fortunately the GFP1 HA Spaghetti Monster RANbody doesn...

...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying... the supplied donor plasmid will express GFP. If there’s GFP expression, presumably the endogenous target...the Madison lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous... easily and seamlessly remove the integrated GFP reporter leaving you with just your desired genomic edit...Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide ...RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP ratio...

...the addition of the enhanced GFP (EGFP)-type termini. mNeonGreen was reported as the brightest monomeric...fluorescent protein. In vitro, mWasabi is 1.6-fold brighter than EGFP and is photostable. It also has narrow ... to 3 times brighter than the most commonly used GFPs and YFPs. Its excitation maxima is at 506 nm and...emission filter. This combination results in a 2.6-fold increase in signal relative to mCerulean imaged ... or for live cell imaging of fusions and FRET reporters. mTFP1 is also insensitive to physiologically ...it does not significantly excite at 400 nm while EGFP does. Find mWasabi plasmids here! One of the most...

...constitutive promoter upstream of GFP increased expression of GFP six-fold, but a stabilized promoter increased...of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a, R6K, and ColE2 plasmids...DNA sequences and have been shown to achieve ~100 fold repression in E. coli. In addition, TALEs bind non-cooperatively...buffered against the effects of copy number on reporter gene expression. On the other hand, using a constitutive...promoter in the same background resulted in a 20-fold variation of gene expression. Because pSC101 is ...increased expression only two fold. Moreover, gene expression levels returned to the baseline after 3 hours...

...: a 24x scaffold fully occupied with GFP has a molecular weight of 1,400 kDa vs 24 kDa for GFP alone. ...-molecule reporter of intracellular processes. Initially, Tanenbaum et al. observed some GFP aggregation...SunTag scaffold. This scaffold contains 10-24 copies of the short epitope GCN4. GCN4 recruits GFP fused...aggregation, which they reduced by using superfolder GFP (sfGFP) with the small solubility tag GB1. In SunTag...nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.   Tanenbaum et al. examined the power...plasma membrane-targeted CAAX-SunTag was 18-fold brighter than sfGFP! The high SunTag signal allowed them to...physiology. Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they ...

... three components: a GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein...protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein...64543or 64542) When the reporter mRNA is first transcribed, it is bound by both the GFP and the RFP proteins...their reporter mRNAs. Using the TRICK plasmids, Halstead et al demonstrated that 91% of reporter mRNAs...customized fluorescent reporters can be challenging enough, but incorporating multiple reporters into a single ...pyralis) reporter plasmids for use in high-throughput screening of pre-mRNA splicing. This reporter system...Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B Mitochondria mito-BFP...

...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... Nano-Lanterns. RLuc8 is 5.5-fold brighter than RLuc but NLuc is 30-fold brighter than RLuc8 (6). Susuki... The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase...compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly...sensitive you can make a reaction. NLuc requires 100-fold less protein to produce the same amount of light...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected/expressing...

... (Avena sativa). In darkness, the AsLOV2 domain folds back on itself in a closed conformation, and in ...Lab monitored how AsLOV2 insertion into mCherry or GFP nanobodies affected binding of their target proteins...co-localization with a membrane-localized mCherry or GFP protein. With this system, if the OptoNB can bind...more OptoNBs, one against mCherry and one against GFP. Insertion of AsLOV2 into loop 1 always generated....  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced ...activated Ras/Erk signaling by using a fluorescent reporter for Erk kinase translocation. These results demonstrate...control protein activity Read about RANbodies, reporter nanobody fusions Learn more about using the secondary...

...of varying strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013...various combinations of protein tags (His, Avi, and GFP) as well as protease sites to facilitate protein ...library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid set 3000 Series Confronted...’ end of the leader sequence. Using a variety of GFP and RFP fusions as their test genes, the researchers... an Orthogonal Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1 (2015): 31-37. pCri...plasmids contain peptides to facilitate protein folding and/or protease sites to remove the epitope tags... pastoris depending upon your specific protein folding needs. The pCri system has recently been used ...

...released Anti-GFP [N86/8R] as another option for targeting GFP. But why might you need a second GFP antibody...PMCID: PMC9015817.   Meet anti-GFP [N86/8R]! by: Ashley Waldron Anti-GFP [N86/38.1R] was among the first...lentiviral Car constructs Anti-GFP [N86/8R] ready-to-use antibody Hyperfolder YFP CRISPR-Csm: A novel RNA...western blot for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page...experiment, consider hfYFP and LSSmGFP.     Fig. 4: hfYFP retains folding and fluorescence in high ...ubiquitous promoter drives expression of an mCherry reporter.  After using mCherry for FACS sorting of the ...?  Similarities:  Generated against full length GFP and converted to recombinant form by the NeuroMab...

...set and have reported brightness measurements. Here, EGFP outperforms the improved folding variants, presumably...imaging with this set are mTagBFP2, EGFP or one of the improved GFP variants, mRuby2 or TagRFP-T, and an...In mammalian cells, one of the improved folding variants of EGFP like mEmerald or Clover is probably best... / em 527 nm) can be imaged together but CFP and GFP (ex 488 nm / em 507 nm) show some crosstalk between...

...S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness... There are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made...examined on a case-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a protonated and...its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles a cylinder...2013; Hilaire et al., 2017). GFP can only be used for tagging proteins GFP fusion proteins are made by ... a fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps... al., 2018). GFP is not tolerated in my fusion It is standard practice to attach the GFP to the N- or ...

...aptamers can either fold to incorporate their target molecule, such as a metal ion, or fold to incorporate ...nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing that GFP’s  structure... Experiments Learn more about GFP Controlling Protein Activity with GFP Resources at Addgene.org Find...highly customizable in terms of how they actually “report” as biosensors. Making aptamers glow The flexibility...increase its fluorescence, essentially mimicking GFP.  Though the aptamer, called Spinach, required longer...Jaffrey lab has since developed new RNA-based scaffolds to contain and stabilize Broccoli cassettes, thereby...

...irreversible PA-FP reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating...mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP to display almost... the anionic form, which allows PA-GFP to exhibit up to a 100-fold increase in fluorescence when illuminated...Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission (nm) PA-GFP Monomer UV-Violet...example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) the Youle Lab was able...illuminated with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent PA-FPs by ...in vivo imaging models perfer the PA-RFPs over PA-GFP. Another class of irreversible PA-FPs are the photoconvertible...

...detects. This change then generates a reporter signal. Reporter signals may be electrochemical or light-based...light-based, with luminescent and fluorescent reporters being especially popular. We’ll give you an introduction...biosensors as an example. Many calcium biosensors include GFP fused to calmodulin (CaM) and the chicken myosin ...calcium-dependent increases in fluorescence of up to 11,000-fold. FRET biosensors are another common class of biosensors...glutamate sensor pCMV(MinDis).iGluSnFR. Picking your reporter Don’t forget about the other end of the biosensor...

...GBPs/GFP. The new system, CRE-DOG (Cre Dependent On GFP), is activated by GFP and derivatives GFP and ...to bind GFP. The idea of using GFP as a scaffold suddenly seemed very realistic. Envisioning GFP as a substrate... of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter lines...a UAS-driven luciferase reporter construct. In the absence of GFP, no reporter output was observed in ...electroporated T-DDOGs, GFP, and a reporter construct into the mouse retina. GFP expression robustly activated...T-DDOGs with two mouse GFP reporter lines; again, TdTomato was seen only in cells with GFP, at a high activation...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted...

... later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the... obtained using ddPCR are generally two to three fold higher than those achieved using the standard qPCR...expression and/or function. These data are sometimes reported or posted on the material page for the corresponding...viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445-AAVrg) alone at 1.1E6 vg/mL, ...prep # 55632-AAVrg). pAAV-CAG- FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from Edward Boyden (Addgene viral...recipient’s initial thaw will be accounted for in our reported titers. Lentiviral vectors are titered using a...

...Yan et al.’s reporter contains 24 copies of SunTag, a synthetic scaffold that can recruit GFP fused to the...following components: a GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein...protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein...64543) Before translation, both GFP and RFP are bound to the reporter, causing it to appear yellow. The...translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the reporter. Because it contains...translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known to be important...correlated with GFP intensity, showing that TRICK is an accurate readout of relative reporter translation....