...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...presence of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted...selectively manipulate only GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies...tested pairs of their GFP-binding proteins (GBPs) to find those that could co-occupy GFP. Once they’d found...culture system. In cells cotransfected with GFP and the GBPs, GFP linked the two GBPs together to create a...

...process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red color was observed...exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP had turned...cytosol-targeted GFP. White circles represent the irradiated area. Photoconverted GFP spreads through ...Applications and limitations of GFP photoconversion One concern with photoconverting GFP is the expression level...intensity increases with GFP concentration and saturates around 30 uM. For low-abundance GFP fusions, a photoconversion...repurpose GFP is great news for microscopy enthusiasts! Addgene has empty backbones containing GFP variants...Since the first research applications of GFP were published in the 1990s, biologists have spent a lot...

...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...expressing GFP from cells that are not. Developmental/transgenic uses: Because of its stability, GFP can be...derivatives are a staple in the lab. GFP is used in research across a vast array of biological disciplines... widespread use in research. Many other mutations have since been introduced to GFP and new iterations...Fluorescent Protein Mutations Relative to Wildtype GFP EGFP F64L; S65T EYFP S65G; V68L; S72A; T203Y mYFP...halves of EGFP undergo folding, maturation, and fluorescence. Biosensors: A wide array of GFP-based fluorescent...from proteins such as Green Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish...

...discovery of GFP The story of how fluorescent proteins like GFP came to be used as research tools is particularly...their work on GFP. This great honor serves to highlight how useful fluorescent proteins like GFP really are...now call GFP, absorbs that blue light and glows green. In the late 1980s, another researcher, Douglas ...started to take notice of GFP, and began to realize its potential as a research tool. Soon, the number of...fluorescent ones, like Green Fluorescent Protein (GFP). These molecules occur naturally in bioluminescent...study the A. victoria gene responsible for encoding GFP, and, in 1992, he reported its sequence. Soon after... Prasher’s collaborator Martin Chalfie expressed GFP in exogenous organisms (E. coli, and later C. elegans...

...Addgene! mRFP-GFP-LC3 tandem fluorescent protein This autophagy biosensor contains mRFP and GFP fused to the...flux. mRFP-GFP-LC3 functions as an autophagy bionsensor via the same mechanism as Rosella: GFP is quenched...decrease in red puncta. Find the mRFP-GFP-LC3 plasmid at Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe ...autophagy biosensor is the GFP-LC3-RFP-LC3ΔG probe. It’s similar to the mRFP-GFP-LC3 Tandem Fluorescent Protein...molecule of GFP-tagged LC3 for every one molecule of RFP-tagged LC3∆G. Like the unmodified LC3, GFP-LC3 is ...Autophagic flux is estimated by the GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence...variant, DsRed.T3, that’s connected to a pH-sensitive GFP variant, super ecliptic pHluorin (SEP). In the absence...

...sender” cells express GFP on the surface and “receiver” cells express an anti-GFP nanobody on the cell ...to track cell-cell interactions By Jennifer Tsang GFP-based Touching Nexus (G-baToN) is a new tool developed...cell surface. When the cells interact, GFP is transferred to the receiver cell, and the receiver cell turns...other fluorophore/antibody pairs. Because the anti-GFP nanobody cross-reacts with BFP, sender cells expressing...expression of cytoplasmic-tdTomato and Synaptophysin-GFP. Synapse targeting GCaMP6s from the Rylan Larsen ... to the repository that will help epigenetic researchers custom design their own engineering plasmid tools...

...switching, TRICK requires the following components: a GFP protein that binds to the coding region of a reporter...reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter...Plasmid 64542 or 64543) Before translation, both GFP and RFP are bound to the reporter, causing it to ...appear yellow. The act of translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the... contains a nuclear localization signal, NLS-PCP-GFP then returns to the nucleus to bind cognate untranslated...verified that translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known...Oskar protein levels were negatively correlated with GFP intensity, showing that TRICK is an accurate readout...

...engineered BFP variant that converts to GFP after a base editing event. GFP positive cells can be sorted and ... their hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains...the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra. Thus...lab was inspired by work that used an integrated GFP reporter that is converted into BFP (blue fluorescent...engineered a BFP variant that undergoes conversion to GFP after being targeted by a cytidine deaminase base... a successful base editing event would result in GFP fluorescence that can be visualized and sorted via...results in shift in emission spectra from BFP to GFP. Image from Standage-Beier et al., 2019. The team...

...al. observed some GFP aggregation, which they reduced by using superfolder GFP (sfGFP) with the small solubility...scaffold fully occupied with GFP has a molecular weight of 1,400 kDa vs 24 kDa for GFP alone. Although traditional...nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.   Tanenbaum et al. examined the power... copies of the short epitope GCN4. GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed...physiology. Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they ...they examined the effects of mitoNEET-SunTag-GFP. As expected, they obtained bright images of mitochondria... gfp...

... the GFP-Puro will be inserted just upstream of a stop codon. One potential concern is if the GFP-Puro...Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month...detailed protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of...microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary...necessary for knock-in: one on either side of the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies...microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the locus (see figure below). ... overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology ...

... of GFP, it's variants, and their relevant mutations, check out Marcy's post from last week on GFP. Additionally... a few hours. For example, superfolder GFP (sfGFP) and mNeonGFP can fold in <10min at 37°C, mCherry takes...are PA-GFP, Dendra2, and the mEOS proteins. Some proteins are reversibly switchable (e.g. rsEGFP, Dreiklang...affected by the temperature. For instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most... the discovery of the Green Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria....below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP (CFP), and yellow...be found at Addgene. Split FPs – some FPs (e.g. GFP, Venus) can be split into two halves, which are non-fluorescent...

...from BRAF-edited and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification... for gene editing. A virus encoding GFP plus a gRNA targeting GFP (pXPR_011, 59702-LV) was used to assay... by the virally-delivered GFP gRNA, will edit out the virally-delivered GFP gene. So, cells with no Cas9...I thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low...expressed GFP) that I couldn’t get a good image. You could probably also do Western blotting against GFP expression...amplification products (GFP-GFP duplex) as a control. 4. After optimizing the reaction and confirming the presence... didn’t. 2. In the original publication they see GFP expression in about 20% of their Cas9-expressing ...

...pAAV.hSyn-FLEX.iGABASnFR.F102G pAAV-Syn-Archon1-KGC-GFP-ER2 pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2] pAAV-FLEX-tdTomato ...(pAAV-Syn-Archon1-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2]) of this...pAAV-mDlx-NLS-mRuby2 CAG-NLS-GFP pAAV-hSyn-mCherry Read on to learn more!  Calcium sensor AAV The GFP-based calcium...37825  AAV8*, AAV9*  pAAV-CAG-GFP 50465  AAV8*, AAV9*  pAAV-hSyn-EGFP *Also coming in our new 20 ...pAAV-GFAP104-mCherry, pAAV-mDlx-NLS-mRuby2, CAG-NLS-GFP,and pAAV-hSyn-mCherry at Addgene. Viral vectors coming...IRES EGFP Chimeric channels for neuronal manipulation 119742  AAV5  AAV SYN PSAM4 GlyR IRES EGFP Chimeric...iGABASnFR and its F102G variant in AAV1 at Addgene! Archon1, a voltage sensor AAV Historically, electrical ...

...respectively of a GFP dependent Cre. The two segments come together in the presence of GFP to form a functional... activation to cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-DOG (75469), is also available...-2 proteins ChR2 for targeted expression DREADDs GFP dependent Cre and FLPo And more!  New lentiviral...in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain GABA-...Karl Deisseroth 83895 AAVrg pAAV-hDlx-Flex-GFP-Fishell_6 Gordon Fishell   Optogenetics Optogenetics...transgenic reporter lines that supply a variety of GFP expression patterns, thus allowing you to further...AAV2 pAAV-Ef1a-fDIO mCherry 83900 AAVrg pAAV-mDlx-GFP-Fishell-1 Biosensors Plasmid Serotype Name ...

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

... structures GFP is limited in its ability to label subcellular structures in the CNS. GFP and other FPs...connectomic tracers Fluorescent proteins such as GFP have been used for a long time as probes to study...fluorescent signals or using anti-FP antibodies. GFP emits light in only a single channel. In multi-color...these FPs is that they are less photostable than GFP. In addition, antibody-based amplification of their..., prone to aggregation and cross-react with anti-GFP antibodies, making them difficult to use in a multicolor...with high fidelity and at lower concentrations than GFP. smFPs enable better labeling of these fine structures...moleculare were better resolved with smFP_FLAG than GFP.   3. Enhanced ability to distinguish weakly expressed...

...to endogenously tag a wide variety of genes with GFP. These plasmids are available through Addgene, and...is the addition of a useful coding sequence (i.e. GFP) into a gene while leaving other features of the ...we show an example gene targeted for tagging with GFP at the C terminus, resulting in one edited allele...allele, or gene copy. The coding sequence for GFP is transcribed and translated along with the gene, producing...shown in the example above, we inserted a linker and GFP tag at the end of the last exon of the gene to ensure...matching the gene of interest, a linker, and the GFP tag sequence to be inserted. Created with BioRender.com...Cas9 makes at the target site. The whole segment—a GFP tag and 1kb of homologous DNA flanking both sides...

...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning and...and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid...human immunodeficiency virus (HIV) genome. For research purposes, retroviral vectors have been developed...Retroviral vectors make up a large toolbox used by researchers for (among other things) gene delivery, generating...empty MuLV vector from the Weinberg lab enables researchers to express siRNAs of their choice in mammalian...

...live cells are GFP+. In this plot, with adjusted voltage, cells that did not receive the GFP protein are ...-case result. For example, you have introduced a GFP protein into your cells (visualized via the FITC ... to be clustered at signals below 103, while the GFP+ population cluster would be shifted to the right...samples and controls were processed the same way, the GFP- population should overlap with the negative control...control population (Figure 1). Based on where the GFP- population ends, you can draw a gate which selects...selects anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the...better interpretation. Priyamvada Prathima has a research background in protein mutagenesis and cancer immunotherapies...

...recombinase. If GFP is under control of an endogenous promoter, you can use expression GFP to track cells...tag an endogenous protein with GFP, as seen in blue flame plasmid OCT4-eGFP-PGK-Puro from the Jaenisch lab...In order to both knockout a given gene and insert GFP into the genome, we’d create a plasmid similar to...to the one shown below, where the sequence of GFP is cloned together with the Neomycin resistance (NeoR... 3 of the targeted gene. Upon recombination, the GFP/NeoR cassette is inserted in place of exon 2. Thus...gene is disrupted (knocked out) but the inserted GFP is expressed (knocked in). As seen in the above example...knockout/knock-in targeting vector designed to insert GFP into a given locus.   Conditional knockouts ...