... 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...our CRISPR guide Find CRISPR plasmids for your research Find resources and software for your CRISPR experiment...

...newly formed proteins.  Limitations with GFP in imaging  While GFP has been an immensely powerful tool to...impossible for scientists to use GFP to image nascent proteins. In addition GFP tags are large, dim, and permanently...proteins in vivo, it does have some limitations. GFP has long protein maturation times which limits its...experiment the Stasevich lab observed colocalization of GFP tagged HA frankenbody with an mCherry, 4xHA tagged...ratio five fold over the 1xHA tag.  Lastly, the researchers found that the HA frankenbody is effective in... be used for imaging in whole organisms. The researchers used the HA frankenbody to successfully track...

... comparison, capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of...AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains they were derived...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...

...about Professor Cepko's recent work on developing GFP-activated proteins, read our recent blog post. Check...Multiple Genes to Cells for Gene Therapy 28:40-35:10 : GFP Activatable Tools Being Developed in the Cepko Lab...C.Y., et al. “Cell type-specific manipulation with GFP-dependent Cre recombinase.” Nat. Neurosci. 18(9) ...on the Addgene Blog Read our Recent Blog Post on GFP Activable Plasmid Tools Being Developed in the Cepko...Medical School researcher Connie Cepko. Listen to the podcast below to hear all about the research being conducted...

...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...only for translational research, such as gene therapy, but also basic research. To improve cell-type specificity...valuable tools for basic as well as translational research for gene therapy. The Roska lab hopes to design...

...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...

...pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR...pSpCas9n(BB)-2A-GFP (PX461) - Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses...requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses...nick, rather than cleave DNA. Find pSpCas9n(BB)-2A-GFP (PX461). pSpCas9(BB)-2A-Puro (PX459) - This mammalian...Browse our CRISPR collection Find plasmids for your research  Learn about our viral service ...

...Serotype Name 105530  AAV1 pAAV.CMV.PI.EGFP.WPRE.bGH 37825  AAV1 pAAV-CAG-GFP 59462  AAV2 pAAV-CAG-tdTomato... testing) pAAV-CAG-GFP 50465 AAV8, 9 (20 µL size serotype testing) AAV-hSyn-EGFP Biosensor AAV The...Plasmid Serotype Name 99039  AAVrg pAAV-CamKII-ArchT-GFP (PV2527) 105679  AAV5 ppAAV-CKIIa-stGtACR1-FusionRed...testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP Recombinases...various serotypes of two popular control AAV encoding GFP. Check out our inventory of serotype testing AAV ...Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP Recombinase AAV Finally, we are excited to add...Serotype Name 119472  AAV5  AV SYN PSAM4 GlyR IRES EGFP   Biosensors Plasmid Serotype Name 105322...

...line expressing a GFP-tagged version of a protein of interest. The binding of the GFP nanobody to its target... proximity to the GFP-tagged protein (albeit slightly offset due to the nanobody-GFP interaction). The...ligase TurboID fused to a conditionally stabilized GFP-nanobody rather than a specific protein of interest...The modularity of the system provides researchers better flexibility and makes the approach more accessible...generate “single-use” transgenic lines. Instead, researchers can generate and maintain transgenic lines that...error rate. For a one kilobase insertion, the researchers reported an editing error rate of less than 0.3%...   This new barcoding technology would allow researchers to avoid the off-target effects of hi-multiplexing...

...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...identified a family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on...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...Cas9 gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy...plasmid undergoes HR or SSA to reconstitute functional GFP, and the cells will turn green. Mashiko et al.,...

...sensor was engineered from a GFP variant and a DA receptor such that GFP fluoresces when the receptor ...on a similar strategy: a GFP variant bound to a DA receptor, such that the GFP fluoresces upon DA-receptor...changes in DA concentration. To address this, researchers from some labs have developed fluorescent-protein... tool to detect and image DA in live animals (Patriarchi et al., 2018). Similar to calcium and glutamate...PMID: 4403305. PubMed Central PMCID: PMC426888. Patriarchi, Tommaso, et al. "Ultrafast neuronal imaging ...protocols and videos Find biosensor AAVs for your research ...

...electron microscopy analysis is time consuming. GFP-tagged Atg8p or LC3: ATG8 is part of a group of genes...with autophagosomal membranes, so tagging it with GFP allows for tracking the localization or accumulation...autophagosomes and autophagic bodies in yeast. A GFP-tagged version of LC3, the mammalian Atg8p homolog...pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP variant, super ecliptic pHluorin (SEP). They are ...Fluorescent Protein Oligomerization Seeing Red: Simple GFP Photoconversion Light Sheet Fluorescence Microscopy...Levine, B. (2010). Methods in Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028...

...TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing GFP that can be...during optimization experiments. This version of GFP will contain a phosphoserine at amino acid 17 instead...instead of a stop codon, which will allow full length GFP to be translated), and second, through a gel shift...protein. Up to 80% p-Ser incorporation into E17TAG GFP has been achieved with the Rinehart system (6); however...bacterial strain – Addgene #68306 pCRT7 NT TOPO E17TAG GFP plasmid - Addgene #68295 SupD plasmid – Addgene #...postdoctoral fellow at the Morgridge Institute for Research in Madison, Wisconsin. It is commonly cited that...perform in vitro kinase reactions. Often, however, researchers lack knowledge of the regulatory kinase, access...

...nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing that GFP’s  structure... Experiments Learn more about GFP Controlling Protein Activity with GFP Resources at Addgene.org Find...increase its fluorescence, essentially mimicking GFP.  Though the aptamer, called Spinach, required longer...or as fluorophores in several different ways. Researchers have used oligonucleotides in fluorescent visualization...

...signal to noise ratio of the system, they modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly...the amount of background fluorescence from unbound GFP-dCas9. With these improvements, labeling efficiency...sequences, which recruit fluorescent proteins BFP, GFP, and RFP fused to the respective hairpin binding ...Fluorescent Proteins Control Protein Activity with GFP Resources on Asddgene.org Find Fluorescent Protein...Colorful CRISPR technologies are helping researchers visualize the genome and its organization within...low resolution and can’t be used in vivo. Some researchers have used fluorescently tagged DNA-binding proteins...CRISPR. Early CRISPR labeling techniques allowed researchers to visualize nearly any single genomic locus,...

...array plus a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted...4 targets when an array of 4 crRNAs, Cpf1, and a GFP tag were transfected on a single plasmid. This is...~75% of neurons were co-transduced with Cpf1 and GFP.  Of these neurons, ~15% had indels at all 3 targeted...lentiviral transduction One vector expresses Cas9, eGFP, and up to 4 gRNAs Each gRNA requires its own promoter... interested in using Cpf1 multiplexing in your research? Check out the Cpf1 multiplexing plasmids available...Multiplexing Resources Find CRISPR Plasmids for Your Research Catch up on Your CRISPR Background with Our Guide...

...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins...Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B Mitochondria mito-BFP...GTPase in division) GFP-Mff (outer membrane protein) Late Endosome mCherry-Rab7A GFP-Rab7A     ...actuator. In 2012, Adam Cohen's lab presented Archaerhodopsin 3 (Arch) as a new rhodopsin based voltage indicator...protein Knock-off, is made up of three components: a GFP protein that binds to the coding region of a reporter...reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter...these dCas9s with one of three fluorescent proteins (GFP, BFP and mCherry), the authors were able to visualize...

...includes a set of plasmids each containing an anti-GFP nanobody fused with a different functional element...using different detection methods. Since the anti-GFP nanobody is able to bind fluorescent proteins derived...here can be used to target any protein fused with GFP, YFP, mCerulean, or other related fluorophores. Find...Arnold  Listen to the MarathonRT podcast segment Researchers from Dr. Anna Pyle’s lab at Yale University recently...them be analyzed by mass spectrometry allowing a researcher to specifically identify proteins that are enriched...

... to track where GFP labelled B cells were in mounted slices of these organs. These GFP labelled cells ...enabled by fluorescent proteins The optimization of GFP and its derivatives by the late Roger Tsien changed...stimulus. Using an epifluorescence microscope and GFP fusion proteins controlled by promoters specific ...the immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph... the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after infection...environments has never been easy and, for many years, researchers lacked good tools to directly follow immune cells...thymus to the lymph nodes. These methods gave researchers much more information regarding immune cell mobility...

...lactose released activates expression of GFP from the lac operon. GFP expression can then be used to determine...(including lactose), and a lac operon controlled GFP. When the enzyme breaks down the HMO of interest,...proteins have enabled scientists to pursue creative research avenues previously unavailable to them. With these...interactions. Beyond these common applications, researchers are finding new ways to apply fluorescent proteins...Roger Tsien played enormous roles in enabling researchers to utilize and develop these tools by making ...fluorescent protein technologies widely available to the research community. To honor their legacy, we recently ...help scientists share their fluorescent protein research with the academic community by funding their travel...