...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...monitor compartment/protein dynamics or evaluate protein turnover rate. In many cases, however, a GFP-tagged...

...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...Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they examined the... copies of the short epitope GCN4. GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed... the effects of mitoNEET-SunTag-GFP. As expected, they obtained bright images of mitochondria without ... gfp...

...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...

... 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 ...

... 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 ...

... 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...

...live cells are GFP+. In this plot, with adjusted voltage, cells that did not receive the GFP protein are ...below 103, while the GFP+ population cluster would be shifted to the right in comparison, say somewhere between...-case result. For example, you have introduced a GFP protein into your cells (visualized via the FITC ...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...easier to identify by comparing peak positions to the base expression. When compared to the control — which...

... 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...

... 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... didn’t. 2. In the original publication they see GFP expression in about 20% of their Cas9-expressing ...BRDN0001146266) ( 76479-LV and 76480-LV) or control GFP gRNA (BRDN0000561167) (80034-LV) were individually...such edits in the cells transduced by the control GFP-gRNA (Figure 4, 5). When the gel was overexposed,...

...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...fluorescence 100-fold compared to other reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains 11 ...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...specifically detects your protein, is species compatible, and doesn’t come with a high background can ...

... the supplied donor plasmid will express GFP. If there’s GFP expression, presumably the endogenous target...RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP ratio...in the genome) and an out-of-frame H2B (histone)-GFP reporter into the genome of mammalian cells.  ...puromycin resistance gene linked to an in-frame H2B-GFP. This visual read out allows for easy screening of... the gene NFE2L2, the Madison lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in... can easily and seamlessly remove the integrated GFP reporter leaving you with just your desired genomic...in the number of endogenous indel edits when compared to unsorted cells (Wen et al., 2018). Thus SRIRACCHA...

...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...background signal (especially for some older split-GFP constructs or certain cell environments). Also consider... In several cases an FP(1-10) fragment may be compatible with multiple FP(11) fragments, even from a different...

...capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of neurons were ...and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains they were derived from. ...also transduced a higher number of neurons overall (GFP+ cells that also stained for a neuronal marker). ...efficiency of AAV-PHP.B versus AAV-PHP.eB in the CNS. A GFP transgene was packaged into either an AAV-PHP.B or...mouse and the level of transduction, measured via GFP expression, was determined 3 weeks later. Table was...efficiency of AAV.9 versus AAV.PHP.S in the PNS. A GFP transgene was packaged into either an AAV.9 or an...

...channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing... a Cre-dependent fluorescent CatCH-GFP reporter. Expression of GFP will only result in cells where Cre-mCherry...mice to test for cell type-specific expression of GFP. Half (113) expressed in retinal cells four weeks...photoreceptors and horizontal cells, and Cre-dependent-GFP expresses in horizontal cells and ganglion cells,...transduced by both AAVs would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired...cones) or cell classes (ex: photoreceptors which encompassed both rods and cones).  Targeted expression was...retina.  Find the AAV synthetic promoter plasmids! Comparing synthetic promoters performance in mouse, monkey...

...reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating Threonine...mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP to display almost...example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) the Youle Lab was able...the neutral to the anionic form, which allows PA-GFP to exhibit up to a 100-fold increase in fluorescence...when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent PA-FPs...in vivo imaging models perfer the PA-RFPs over PA-GFP. Another class of irreversible PA-FPs are the photoconvertible...Fluorescence) Brightness Excitation/ Emission (nm) PA-GFP Monomer UV-Violet Dark/ Green ~200 Medium 504/ 517...

...the limitations is the size of GFP: ~240 amino acids or about 28 kDa. GFP and its homologues have a beta-barrel...and shape matters The green fluorescent protein (GFP) from Aequorea victoria and its variants are genetically...interference is known as steric hindrance. For instance, GFP may occlude catalytic sites or obstruct binding sites...post-translational modifications. Despite these potential issues, GFP has been used successfully in countless fusion proteins... an inert light emitting module, any tendency of GFP to homodimerize should be avoided because it will...the linker sequence short In the early days of GFP applications, many were concerned with steric hindrance...fluorescent proteins derived from Aequorea victoria GFP sticks out and can be considered a linker (figure...

...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 ...depiction of chromatin within a nucleus -> DNA compaction around histones -> Examples of histone-modifying...The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap cloning systems to generate...