...and best solution is to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald...problematic, several reportedly monomeric FPs are not actually monomeric, at least in practical terms. ...FPs, are not necessarily monomeric, even if they were described as monomeric in a publication. The gold....A., et al., Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science...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...

...reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating Threonine...Emission (nm) PA-GFP Monomer UV-Violet Dark/ Green ~200 Medium 504/ 517 PS-CFP2 Monomer UV-Violet Cyan...mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP to display almost... 583/ 593 mKikGR Monomer UV-Violet Green/ Red 560 High 580/ 591 Dendra2 Monomer UV-Violet or blue ... (nm) Dronpa Monomer UV-Violet Dark/Green N/A (high) High 503/ 518 Padron Monomer Blue Dark/ Green...503/ 518 rsCherry Monomer Yellow Dark/ Red 7 Low 572/ 610 rsCherryrev Monomer Blue Dark/ Red 20 Low.... Both variants are more monomeric than mEos2 (mEos4b is completely monomeric) and display bright and ...

...the enhanced GFP (EGFP)-type termini. mNeonGreen was reported as the brightest monomeric green or yellow...plasmids here! mTFP1: a monomeric teal fluorescent protein mTPF1 is a monomeric version of the tetrameric...bright, monomeric green fluorescent protein. In vitro, mWasabi is 1.6-fold brighter than EGFP and is photostable... to 3 times brighter than the most commonly used GFPs and YFPs. Its excitation maxima is at 506 nm and... things about these proteins is that they are monomeric fluorescent proteins. Fluorescent proteins that...Ai, Hui-wang, et al. "Directed evolution of a monomeric, bright and photostable version of Clavularia ...PMC1698604. Ai, Hui-wang, et al. "Hue-shifted monomeric variants of Clavularia cyan fluorescent protein...

...miRFPs are monomeric and bright Recent series of enhanced monomeric NIR FPs named miRFPs (monomeric infrared... 127 Monomer 394 4.5 37 miRFP709 https://www.addgene.org/79989/ 683 709 78,400 5.4 69 Monomer 192 ...112 Monomer 54 4.5 15  15 miRFP https://www.addgene.org/108409/ 674 703 92,400 9.7 145 Monomer ND ...127 Monomer 700 4.5 90 miRFP713 https://www.addgene.org/136559/ 690 713 99,000 7.0 122 Monomer 980...95,000 10.8 166 Monomer 545 3.7 62 8 miRFP670nano3 645 670 129,000 18.5 387 Monomer 675 4.2 250 9 ...704 93,000 9.9 149 Monomer 1265 4.1 81 miRFP718nano 690 718 79,000 5.6 72 Monomer 885 3.8 33 State-of-the-art... state-of-the-art NIR FPs can be used similar to GFP-like probes by supplying a single gene to cells. ...

... 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...SRIRACCHA plasmids at Addgene GEMCherry2, an improved monomeric fluorescent protein for assessing CRISPR/Cas9 ...

...Expression PA-GFP 504 517 14 Monomer (A206K) pPAGFP-N1 - Mammalian Expression pPAGFP-C1 - Mammalian Expression...Expression pRSETA-PAGFP - Bacterial Expression tandem PA-GFP-KanR - Yeast Expression pFA6a-PA-GFP-kanMX6 - Yeast...Expression pFA6a-link-yEPAGFP-CaUra3 - Yeast Expression mPA-GFP-N1 - Mammalian Expression mPA-GFP-C1 - Mammalian...Protein Fusions with mOX FPs Blog: When is a Monomer not a Monomer? Background Fluorescent proteins are genetically...then scientists have engineered numerous GFP-variants and non-GFP proteins that result in a diverse set ...Proteins that are derived from GFP and contain the A206K mutation are monomeric at all concentrations, so ... 385 448 17 Monomer (A206K) moxBFP - Mammalian Expression mKalama1 385 456 16 5.5 Monomer (A206K) pBad-mKalama1...

...et al. developed monomeric FP timers with less propensity to aggregate. These monomeric FP timers (mK-GO... a given timer in many experimental settings. Monomeric fluorescent protein timers Since the production...et al.) were derived from previously developed monomeric fluorescent proteins mKO and mCherry, respectively...events occurring at varied time scales.  Table 1: Monomeric FP Timers and Associated Plasmids from Subach ...neuroendocrine cells revealed by newly developed monomeric fluorescent timer protein." Molecular biology ... PMID: 11408473. 4. Subach, Fedor V., et al. "Monomeric fluorescent timers that change color from blue...Fluorescent Protein Aggregation Catch Up On Your GFP Basics Get Advice on Choosing Your Fluorescent Protein...

...Peptide-assisted genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic gene..., Myc and SV40 Nuclear Localization Signals, and GFP. B) Workflow to assess Cas9-PAGE editing with viral...PMID: 37095348. Breaking up is hard to do: Monomeric StayGold variants By Mike Lacy In 2022, Atsushi...applications. Several labs have been working to produce monomeric versions that break up the dimerization without...Piatkevich and colleagues generated yet another monomeric StayGold variant (Piatkevich et al., 2023). Check...2941917/v1.  Ivorra-Molla, E., et al. (2023). A monomeric StayGold fluorescent protein. Nature Biotechnology...Piatkevich, K., et al. (2023). Bright and stable monomeric fluorescent proteins derived from StayGold. ResearchSquare...

...S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness...generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen Chromophore... 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...

...the limitations is the size of GFP: ~240 amino acids or about 28 kDa. GFP and its homologues have a beta-barrel...potential issues, GFP has been used successfully in countless fusion proteins. Choose monomeric proteins and...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... an inert light emitting module, any tendency of GFP to homodimerize should be avoided because it will...Keep 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...

...a gene of interest fused to the N-terminus of monomeric Cerulean mCerulean C1 Cyan Mammalian Express...a gene of interest fused to the C-terminus of monomeric Cerulean mTurquoise2 Cyan Mammalian Constructs...a gene of interest fused to the N-terminus of monomeric Venus mVenus C1 Yellow Mammalian Express a gene... gene of interest fused to the C-terminus of monomeric Venus pCEP4YPet-MAMM Yellow Mammalian Expresses...SYFP2 Clover Green Mammalian Expresses Clover (a GFP variant) commonly used with mRuby2 pLSSmOrange-...

...predictable and precise deletion products compared to monomeric Cas9’s. Read the paper in Nature Communications...plasmids Tagging human transcription factors with GFP Kevin White’s lab recently deposited a set of CRISPR...plasmids to tag human transcription factors with GFP. As part of the Encyclopedia of DNA Elements (ENCODE...

...your hard work!   Fluorophores When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent...Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts in your experiments...

... true monomer in cells (Constantini, 2012), whereas it was previously concluded to be monomeric based ...to declare that it is monomeric. The claim that a fluorescent protein is monomeric without the accompanying... can be compared with mEGFP or mTurquoise2, which are established true monomeric fluorescent proteins....studies have used the OSER assay to demonstrate monomeric behaviour in cells (Shaner et al, 2013; Bindels...mTurquoise2 and dTomato, which are established monomeric and dimerizing FPs respectively, are used as positive...which is good evidence that mScarlet-I acts as a monomer in the cell type tested. To conclude, we recommend...fluorescent protein. In the jellyfish Aequorea victoria, AvGFP forms a homodimer. In corals, the red fluorescent...

...-selection) tools used in cloning. 8. Bright Monomeric Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi... PS: they’re free. 3. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection by ...Joachim Goedhart shares some strategies for generating GFP fusion proteins. From considering protein size and...

...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...

...to Amber via a 32 amino acid linker mGFP-10-sREACh-N3 Monomeric EGFP attached to super-REACh via a 10 amino...a gene of interest fused to the N-terminus of monomeric Cerulean mCerulean C1 Cyan Mammalian Express a...a gene of interest fused to the C-terminus of monomeric Cerulean mTurquoise2 Cyan Mammalian Constructs...a gene of interest fused to the N-terminus of monomeric Venus mVenus C1 Yellow Mammalian Express a gene...gene of interest fused to the C-terminus of monomeric Venus pCEP4YPet-MAMM Yellow Mammalian Expresses mammalian...SYFP2 Clover Green Mammalian Expresses Clover (a GFP variant) commonly used with mRuby2 pLSSmOrange-N1...

...conformation.  Nanobodies are a small (~15 kDa), single monomeric variable heavy chain antibody domain.  How do...Lab monitored how AsLOV2 insertion into mCherry or GFP nanobodies affected binding of their target proteins...co-localization with a membrane-localized mCherry or GFP protein. With this system, if the OptoNB can bind...more OptoNBs, one against mCherry and one against GFP. Insertion of AsLOV2 into loop 1 always generated....  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced ...

... 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...

...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...chromophore formation (abundant in eukaryotes) Bright monomeric near-infrared fluorescent proteins as tags and...background signal (especially for some older split-GFP constructs or certain cell environments). Also consider...