...determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below for a possible...Data Figure 1: 293T cells were transfected with the GFP-expression plasmid pRosetta using μg total DNA to...efficiencies as measured by the highest proportion of GFP positive cells without limiting cell growth. Left...Left panels: bright field images; right panels: GFP channel images. Last reviewed on: August 2, 2023...

...determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below for a possible...

...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...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...derivatives are a staple in the lab. GFP is used in research across a vast array of biological disciplines...disciplines and scientists employ GFP for a wide number of functions, including: tagging genes for elucidating...

...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 ...

...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...

...laboratory. The discovery of GFP The story of how fluorescent proteins like GFP came to be used as research...their work on GFP. This great honor serves to highlight how useful fluorescent proteins like GFP really are...fluorescent ones, like Green Fluorescent Protein (GFP). These molecules occur naturally in bioluminescent... calcium ions, and the second, which we now call GFP, absorbs that blue light and glows green. In the ...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...work, scientists really started to take notice of GFP, and began to realize its potential as a research...

...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...

...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...western blot for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page...system Heat-inducible lentiviral Car constructs Anti-GFP [N86/8R] ready-to-use antibody Hyperfolder YFP ...?  Similarities:  Generated against full length GFP and converted to recombinant form by the NeuroMab...variable domains that recognize different epitopes on GFP Monoclonal antibodies can yield relatively low signal... details. Image credit: Addgene.   Find anti-GFP [N86/8R] here!   Andrews NP,  et. al. A toolbox of...

...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...

...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...

... 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...

...pAAV.hSyn-FLEX.iGABASnFR.F102G pAAV-Syn-Archon1-KGC-GFP-ER2 pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2] pAAV-FLEX-tdTomato pAAV-GFAP104...pAAV-mDlx-NLS-mRuby2 CAG-NLS-GFP pAAV-hSyn-mCherry Read on to learn more!  Calcium sensor AAV The GFP-based calcium...pAAV-Syn-Archon1-KGC-GFP-ER2) and cre-dependent version (pAAV-Syn-FLEX-rc [Archon1-KGC-GFP-ER2]) of this membrane-embedding...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...

...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 ...

... have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP nanobodies  stain poorly when tagged with...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... sequences that targeted the following antigens: GFP, RFP,  the histone protein H2A2B, and the actin-binding...recognize the scaffold and is useful for detecting GFP. Spaghetti Monster tagged RANbodies can be detected...expensive Can only stain with one HRP-RANbody at a time GFP, RFP, H2A2B, Gelsolin Spaghetti Monster: HA (H)...Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B Myc Tagged RFP, H2A2B Chicken IgY ...

...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...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...

...self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP...apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11) to increase fluorescence... reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains 11 β-strands and one central ɑ-helix...Lab used FlipGFP to track the spatiotemporal dynamics of apoptosis during embryo development. GFP fluorescence...protein-protein interaction sensor based on tripartite split-GFP association." Scientific reports 3 (2013): 2854. ...PMC2117903. To, Tsz-Leung, et al. "Rational design of a GFP-based fluorogenic caspase reporter for imaging apoptosis...fluorogenic protease reporter by flipping a beta strand of GFP for imaging apoptosis in animals." Journal of the...

...antibodies (mouse, rabbit, and chicken) to this tag! GFP  GFP is more than a peptide – it’s a 238 amino acid...localization. If you need monoclonal GFP antibodies or guidance on designing a GFP tag Addgene has you covered!...acid protein that can also be used as a tag! GFP was originally derived from jellyfish (Aequorea victoria...epitope tag as well as a fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry...antibodies also exist for imaging. The obvious con of GFP is its size – which can possibly interfere with protein...