...puromycin, hygromycin) and fluorescent protein reporters (GFP, BFP, RFP, no FP). The vectors each contain...SB transposase, SB100X, that is approximately 100-fold more efficient than the first-generation transposase...region of the RPL41 gene, to drive the selection/reporter parts of each construct. This new tool kit allows...

...popular studies involve using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following...from the resin and used for various binding or folding assays. Yeast vectors There are several types of...animals, gene targeting and genome modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g... allow you to visualize YGOI in vivo (try pcDNA3-EGFP). If you can get away with generating transiently...

...phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly deactivates...highest level of “output” signal, was only about 10-fold, precluding the study of gene expression at very...leakiness and increased dynamic range to around 100-fold. These improvements will allow researchers to better...

...its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether it interacts...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...genes as reporters and some that repress instead of activate transcription. The HIS3 reporter system (...co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing...referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI...Gal4 activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including...to have Y2H systems that avoid transcriptional reporters altogether; In the split-luciferase system, intein-mediated...

...fusion protein molecules fail to correctly fold? Misfolded FPs do not fluoresce. A dark population is ...disrupt FP folding rendering the protein non-fluorescent. Unfortunately, accumulated misfolded and nonfunctional...Lindsey M. Costantini, and Erik L. Snapp. "Superfolder GFP is fluorescent in oxidizing environments when...potentially affect function [5]. We and others have reported that FP cysteine residues form inappropriate disulfide...been a pervasive view that most FPs in a cell will fold and that the fluorescence pattern is equivalent ...brighter FP, but rather because of the correct folding and fluorophore formation by most, if not all, ...missed the impact of dark, non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments...

...region of a FLAG-binding scFv onto a well-folded scFv scaffold to improve its stability in cells, and use...-FLAG scFv and a stable scFv scaffold. (b) Schematic of anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear...nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets with nuclear (top, 4x-FLAG-mCherry-H2B...induce high levels of gene activation for both reporter and endogenous genes. It is highly specific in...This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and HaloTag fusions,...fusions, while the anti-HA frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown...gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy...identified a family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on...mutagenesis libraries or to insert heterologous genes, reporters, or other markers into a target genome. They did...and demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the...enabled scientists to engineer a wide variety of GFPs with diversity in fluorescence brightness, intensity...

...stage-specific promoter and design a UAS reporter construct, the reporter will only be expressed where and/or...UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter with...QUAS driven expression by ~3,300-fold in Drosophila cells and ~24,000-fold in human HeLa cells as compared...Church lab uses this system to control expression of GFP.)  To turn this inducible system into a repressible...

...following sucrose treatment. Moreover, several reports have shown that cells treated with sucrose or other...of sucrose improve AAV2 yields approximately 1.8-fold without affecting cell viability. This effect appears...endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe any difference...

...Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald, CFP, YFP, BFP, Cerulean...contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies ...in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently...designated mGFP, mVenus, etc. A word of caution is needed because FPs not derived from GFP, i.e. the red...However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution...

...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...a gRNA cassette. It can be used to produce a ~10-fold higher titer of lentiviral particles than the original...together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging studies...

...poor folding and thermal instability at 37°C (Ouellet et al., 2016) and requires a tRNA scaffold to improve...aptamers able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...Broccoli increases its fluorescence by about 1.8-fold, and may be a better choice for imaging than the...complex and the entire aptamer can hybridize and fold. (Alam et al., 2017). Yellow fluorescent aptamers...growth as RNA biology, we are looking forward to reports on the next additions to aptamer soup! References...that was more thermally stable, exhibited less misfolding, and was consequently brighter than Spinach. ... Matthew D. Disney, and Samie R. Jaffrey. "A superfolding Spinach2 reveals the dynamic nature of trinucleotide...

...Chang-Deng Hu Green sfGFP Split super-folder GFP, our most-requested GFP(1-10) and GFP(11) Versatile protein...Commun. 2016 Bo Huang spGFP Split superpositive GFP Split-superpositive GFP reassembly is a fast, efficient...base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles linked below only provide...Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with... Kamiyama Cerulean Cerulean(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with... (abundant in eukaryotes) A Near-Infrared BiFC Reporter for In Vivo Imaging of Protein-Protein Interactions...green-yellow or orange-red fluorogen A split fluorescent reporter with rapid and reversible complementation. Tebo...

...ACE) in real-time. The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry...maximum is reported as 603 nm, while the emission maximum is 675 nm. mCarmine is monomeric, 4-5 fold brighter... ACE system: A versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9...editing tools, the Reuben Harris lab has deposited a reporter plasmid that allows for rapid fluorescence-based... out a frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply...containing rat APOBEC1. Finally, they show that the ACE reporter can also quantify Cas9 cleavage, thus exhibiting...editing activities. Click here to find the ACE reporter plasmid! Martin et al., Nucleic Acids Research...

...molecular brightness (Table 1).  NIR reporters and biosensors NIR reporters can tell us about cellular events...normalization to fluorescence of co-transfected EGFP. e Originally reported as a monomer16, IFP2.0 was later found... state-of-the-art NIR FPs can be used similar to GFP-like probes by supplying a single gene to cells. ...crosstalk-free three-color imaging with green and red GFP-like FPs (Shcherbakova et al., 2016), as well as ...higher apparent cellular brightness than previously reported widely used dimeric iRFPs (Table 1). In general...protein-protein interactions, we recommend using NIR split reporters based on miRFPs (Shcherbakova et al., 2016), since...E., Sivanesan, D. & Michnick, S.W. An infrared reporter to detect spatiotemporal dynamics of protein-protein...

...phosphatase-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked mCherry...elements and combined it with a variety of reporters (i.e. eGFP) and effectors (i.e. DREADDs). The team ...are available at Addgene. Kim, et al. Stem Cell Reports. 2015. PubMed PMID: 25772473 Targeting and manipulating...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...off-target cutting, and they found that HF-BE3 showed 37-fold less off-target editing than BE3, with only a slight...reagents shared via Addgene. Narayan S, et al. Cell Reports. 2017. PubMed PMID: 28813671 Listen to the podcast...along with EGFP while retaining an emission spectrum that is easily separable from that of EGFP. This makes...

..., Bernach plans to use a suite of fluorescent reporters. Bacteria containing unstable fluorescent proteins...colonizer, labelled with a constitutively fluorescent reporter can also be used to track how one population responds...using TIMER and an unstable fluorescent protein reporter under the control of promoters that control cell...fluorescent protein topics page Get the basics on GFP Find microbiology blog posts Resources on Addgene.org... the TIMER growth rate bioreporter. During protein maturation, the bioreporter spontaneously changes from...decreased in protein abundance, possibly due to misfolding. But measuring variants one at a time is time...variant library is monitored by fusing the variants to eGFP followed by an internal ribosomal entry site and...

...of posts explains how to critically assess the reported properties of fluorescent proteins, how to do ...protein with a high intrinsic brightness that does not fold well or cannot be detected in a microscopy setup...experimental conditions into account. In particular, the folding and maturation efficiency of the fluorescent protein... QY (EC*QY, sometimes normalized to the value of EGFP). The higher the number, the higher the theoretical...

... strain transformed with pBLADE expressing superfolder GFP instead of mCherry under the PBAD promoter....the DNA-binding domain of AraC using a mCherry reporter downstream of a PBAD promoter. While none of the...contains BLADE, the PBAD promoter, and a mCherry reporter downstream of the PBAD promoter. To use this plasmid...

..., Hess et al. successfully evolved wild type GFP to EGFP using CRISPR-X and subsequent FACS sorting. They...-ome using a quick, affordable, and ubiquitous reporter assay. In their recent publication, Kroeze et ...with unique tissue-specific promoters expressing GFP, tetracycline response elements, and shRNAs many ... different human cell models and identified ~4-5-fold more fitness-related genes than have been found ... of DsRed connected to a pH-sensitive variant of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The...when it is difficult for a cell to synthesize or fold a protein because of its length. Mark Howarth’s ...endogenous proteins. As described in their recent Cell Reports paper, the Kanemaki lab overcame this hurdle using...