...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...Use this protocol to generate lentivirus Protocols...downstream applications such as stable-cell line generation. Workflow Timeline Day 0: Seed 293T packaging...adherent cells) 0.45 μm polyethersulfone filter, Nalgene, 565-0010 Microcentrifuge tubes, Neptune 3745.X...

...Transfections allow for transient expression of a gene of interest in a target cell line and can be useful...determine what ratio gives the highest percentage of GFP positive cells. Refer to the table below for a possible... Protocols General Transfection General Transfection You may also like... Viral...Service Introduction This protocol describes a general method for transfecting mammalian cells using linear...

...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...co-opted to control gene expression, he could then selectively manipulate only GFP-labeled cells. Once Tang ...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...repurpose GFP is great news for microscopy enthusiasts! Addgene has empty backbones containing GFP variants...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...have created a transgenic GFP-containing organism,” says Hanson, “using a gene encoding one of the unexpectedly...

...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...and scientists employ GFP for a wide number of functions, including: tagging genes for elucidating their...Placing GFP under the control of a promoter of interest can be used to effectively monitor gene expression...type may have the GFP under the control of an additional promoter from that of the gene of interest, or ...expressing GFP from cells that are not. Developmental/transgenic uses: Because of its stability, GFP can be...implantation and generation of transgenic mice. Purification: GFP can be used as a general epitope tag for...scientists began engineering new versions of GFP through mutagenesis in order to improve its physical and biochemical...

...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...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...

...laboratory. The discovery of GFP The story of how fluorescent proteins like GFP came to be used as research...He began to study the A. victoria gene responsible for encoding GFP, and, in 1992, he reported its sequence...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 ...new green glowing protein could be used to measure gene transcription, as well as to track protein localization... Prasher’s collaborator Martin Chalfie expressed GFP in exogenous organisms (E. coli, and later C. elegans...

...decrease in red puncta. Find the mRFP-GFP-LC3 plasmid at Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe ...flux. mRFP-GFP-LC3 functions as an autophagy bionsensor via the same mechanism as Rosella: GFP is quenched...autophagy biosensor is the GFP-LC3-RFP-LC3ΔG probe. It’s similar to the mRFP-GFP-LC3 Tandem Fluorescent Protein...Autophagic flux is estimated by the GFP/RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence...the mitochondria-targeted Keima variant at Addgene! mRFP-GFP-LC3 tandem fluorescent protein This autophagy...translation, this fusion protein is cleaved to generate one molecule of GFP-tagged LC3 for every one molecule of...decrease in autophagic flux.   Find the GFP-LC3-RFP-LC3∆G plasmid at Addgene! References Klionsky, Daniel J....

... at Addgene. Additional Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background...sites on the nascent polypeptide recruit scFV-GFP, generating green puncta that colocalize with the red reporter...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...

...released Anti-GFP [N86/8R] as another option for targeting GFP. But why might you need a second GFP antibody...on the Anti-GFP [N86/8R] page for more details. Image credit: Addgene.   Find anti-GFP [N86/8R] here...were compared head-to-head for gene expression changes of non-target genes and RNA-seq of a nuclear (MALAT1...PMCID: PMC9015817.   Meet anti-GFP [N86/8R]! by: Ashley Waldron Anti-GFP [N86/38.1R] was among the first...antibody?  Similarities:  Generated against full length GFP and converted to recombinant form by the NeuroMab...system Heat-inducible lentiviral Car constructs Anti-GFP [N86/8R] ready-to-use antibody Hyperfolder YFP ...variable domains that recognize different epitopes on GFP Monoclonal antibodies can yield relatively low signal...

... their hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains...engineered BFP variant that converts to GFP after a base editing event. GFP positive cells can be sorted and ...without relying solely on GFP (Katti et al., 2020). They named this reporter “Gene On” or GO. GO works by...changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra...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...

...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...used to improve CRISPR-based activation of target genes, but we’ll save that application for another day...

...the most efficient for gene editing. A virus encoding GFP plus a gRNA targeting GFP (pXPR_011, 59702-LV)...by the virally-delivered GFP gRNA, will edit out the virally-delivered GFP gene. So, cells with no Cas9... BRAF-edited and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification... to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in ...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...Targeting different regions of the gene is also useful in case the gene has a weird mutation that you didn...

... the GFP-Puro will be inserted just upstream of a stop codon. One potential concern is if the GFP-Puro...ready-made PITCh plasmids available from Addgene are perfect for expressing GFP from a given promoter, and the ...harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh (Precise Integration into Target ...Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and...than that observed with NHEJ. PITCh: Using MMEJ for gene knock-in Building on the lab’s previous work, Sakuma...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...

... 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...these biosensors can be found at Addgene. Split FPs – some FPs (e.g. GFP, Venus) can be split into two ...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...

...respectively of a GFP dependent Cre. The two segments come together in the presence of GFP to form a functional...supply a variety of GFP expression patterns, thus allowing you to further restrict gene activation to cells...cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-DOG (75469), is also available in AAV1. ...AAVrg pAAV-hDlx-Flex-GFP-Fishell_6 Gordon Fishell   Optogenetics Optogenetics continues to supply neuroscience...-2 proteins ChR2 for targeted expression DREADDs GFP dependent Cre and FLPo And more!  New lentiviral...inhibition library that inhibits over 18,000 human genes and is used for genome-wide inhibition screening...in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain GABA-...

...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...mCherry, pAAV-mDlx-NLS-mRuby2, CAG-NLS-GFP,and pAAV-hSyn-mCherry at Addgene. Viral vectors coming soon! These...37825  AAV8*, AAV9*  pAAV-CAG-GFP 50465  AAV8*, AAV9*  pAAV-hSyn-EGFP *Also coming in our new 20 ...variant of jGCaMP7 in AAV1 at Addgene! See all calcium sensor AAV at Addgene. GABA sensor AAV GABA is the...IRES EGFP Chimeric channels for neuronal manipulation 119742  AAV5  AAV SYN PSAM4 GlyR IRES EGFP Chimeric...

... 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...Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells...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...best solution is to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald, CFP...However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution...noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could be forming...

... Figure 1: Gene editing is the addition of a useful coding sequence (i.e. GFP) into a gene while leaving...features of the gene intact. Here, we show an example gene targeted for tagging with GFP at the C terminus...allele, or gene copy. The coding sequence for GFP is transcribed and translated along with the gene, producing...tag a wide variety of genes with GFP. These plasmids are available through Addgene, and the stem cell lines..., we inserted a linker and GFP tag at the end of the last exon of the gene to ensure it would be transcribed... kb homology arms matching the gene of interest, a linker, and the GFP tag sequence to be inserted. Created...insertion of the GFP tag precisely where we need it, in this example, at the C-terminus of the gene of interest...