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We narrowed to 46 results for: Folding Reporter GFP

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  1. FlipGFP, a novel fluorescence protease reporter to study apoptosis

    Type
    Blog Post
    Published
    May 21, 2019, 12:10 p.m.
    ...fluorescence 100-fold compared to other reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains 11...apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11) to increase...self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP...fluorescent executioner caspase reporters. Several apoptosis reporters have been developed, such as the...transfer (FRET)-based executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase...FlipCherry: Red fluorescence protease reporter To add to the fluorogenic reporter toolkit, the Shu Lab also designed...range and high brightness. This reporter achieves approximately 100-fold increase in fluorescence after cleavage...

  2. Fluorescent Proteins 101: Green Fluorescent Protein (GFP)

    Type
    Blog Post
    Published
    May 15, 2014, 3:33 p.m.
    ...halves of EGFP undergo folding, maturation, and fluorescence. Biosensors: A wide array of GFP-based fluorescent...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a promoter of interest.... This type of transcription reporting was among the earliest uses of GFP. Fluorescence resonance energy...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...from proteins such as Green Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish...

  3. RANbodies: Reporter Nanobody Fusions

    Type
    Blog Post
    Published
    April 10, 2018, 12:56 p.m.
    ... have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP nanobodies  stain poorly when tagged with...detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties: HRP, Spaghetti...independent of the reporter. The only caveat is that HA staining is weaker than reporter staining. Picking...Picking the right reporter for your RANBody While all three reporters stain cultured cells and tissue sections...Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection Method Uses Pros Cons Antigens...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...

  4. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    Published
    May 26, 2020, 1:15 p.m.
    ...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying... the supplied donor plasmid will express GFP. If there’s GFP expression, presumably the endogenous target...the Madison lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous... easily and seamlessly remove the integrated GFP reporter leaving you with just your desired genomic edit...Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide ...RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP ratio...

  5. Bright Monomeric Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi

    Type
    Blog Post
    Published
    April 25, 2019, 3:01 p.m.
    ...the addition of the enhanced GFP (EGFP)-type termini. mNeonGreen was reported as the brightest monomeric...fluorescent protein. In vitro, mWasabi is 1.6-fold brighter than EGFP and is photostable. It also has narrow ... to 3 times brighter than the most commonly used GFPs and YFPs. Its excitation maxima is at 506 nm and...emission filter. This combination results in a 2.6-fold increase in signal relative to mCerulean imaged ... or for live cell imaging of fusions and FRET reporters. mTFP1 is also insensitive to physiologically ...it does not significantly excite at 400 nm while EGFP does. Find mWasabi plasmids here! One of the most...

  6. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

    Type
    Blog Post
    Published
    Sept. 4, 2018, 12:53 p.m.
    ...constitutive promoter upstream of GFP increased expression of GFP six-fold, but a stabilized promoter increased...of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a, R6K, and ColE2 plasmids...DNA sequences and have been shown to achieve ~100 fold repression in E. coli. In addition, TALEs bind non-cooperatively...buffered against the effects of copy number on reporter gene expression. On the other hand, using a constitutive...promoter in the same background resulted in a 20-fold variation of gene expression. Because pSC101 is ...increased expression only two fold. Moreover, gene expression levels returned to the baseline after 3 hours...

  7. SunTag and Fluorescent Imaging

    Type
    Blog Post
    Published
    March 28, 2017, 2:30 p.m.
    ...: a 24x scaffold fully occupied with GFP has a molecular weight of 1,400 kDa vs 24 kDa for GFP alone. ...-molecule reporter of intracellular processes. Initially, Tanenbaum et al. observed some GFP aggregation...SunTag scaffold. This scaffold contains 10-24 copies of the short epitope GCN4. GCN4 recruits GFP fused...aggregation, which they reduced by using superfolder GFP (sfGFP) with the small solubility tag GB1. In SunTag...nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.   Tanenbaum et al. examined the power...plasma membrane-targeted CAAX-SunTag was 18-fold brighter than sfGFP! The high SunTag signal allowed them to...physiology. Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they ...

  8. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ... three components: a GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein...protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein...64543or 64542) When the reporter mRNA is first transcribed, it is bound by both the GFP and the RFP proteins...their reporter mRNAs. Using the TRICK plasmids, Halstead et al demonstrated that 91% of reporter mRNAs...customized fluorescent reporters can be challenging enough, but incorporating multiple reporters into a single ...pyralis) reporter plasmids for use in high-throughput screening of pre-mRNA splicing. This reporter system...Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B Mitochondria mito-BFP...

  9. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

    Type
    Blog Post
    Published
    Nov. 19, 2019, 2:08 p.m.
    ... (Avena sativa). In darkness, the AsLOV2 domain folds back on itself in a closed conformation, and in ...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 ...activated Ras/Erk signaling by using a fluorescent reporter for Erk kinase translocation. These results demonstrate...control protein activity Read about RANbodies, reporter nanobody fusions Learn more about using the secondary...

  10. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    Published
    Feb. 8, 2018, 12:17 p.m.
    ...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... Nano-Lanterns. RLuc8 is 5.5-fold brighter than RLuc but NLuc is 30-fold brighter than RLuc8 (6). Susuki... The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase...compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly...sensitive you can make a reaction. NLuc requires 100-fold less protein to produce the same amount of light...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected/expressing...

  11. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    Published
    Sept. 14, 2016, 2:30 p.m.
    ...of varying strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013...various combinations of protein tags (His, Avi, and GFP) as well as protease sites to facilitate protein ...library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid set 3000 Series Confronted...’ end of the leader sequence. Using a variety of GFP and RFP fusions as their test genes, the researchers... an Orthogonal Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1 (2015): 31-37. pCri...plasmids contain peptides to facilitate protein folding and/or protease sites to remove the epitope tags... pastoris depending upon your specific protein folding needs. The pCri system has recently been used ...

  12. Hot Plasmids - January 2023

    Type
    Blog Post
    Published
    Jan. 31, 2023, 2:15 p.m.
    ...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...lentiviral Car constructs Anti-GFP [N86/8R] ready-to-use antibody Hyperfolder YFP CRISPR-Csm: A novel RNA...western blot for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page...experiment, consider hfYFP and LSSmGFP.     Fig. 4: hfYFP retains folding and fluorescence in high ...ubiquitous promoter drives expression of an mCherry reporter.  After using mCherry for FACS sorting of the ...?  Similarities:  Generated against full length GFP and converted to recombinant form by the NeuroMab...

  13. Choosing Your Fluorescent Proteins for Multi-Color Imaging

    Type
    Blog Post
    Published
    Oct. 9, 2014, 3 p.m.
    ...set and have reported brightness measurements. Here, EGFP outperforms the improved folding variants, presumably...imaging with this set are mTagBFP2, EGFP or one of the improved GFP variants, mRuby2 or TagRFP-T, and an...In mammalian cells, one of the improved folding variants of EGFP like mEmerald or Clover is probably best... / em 527 nm) can be imaged together but CFP and GFP (ex 488 nm / em 507 nm) show some crosstalk between...

  14. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...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 ...

  15. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    Published
    April 11, 2017, 2:30 p.m.
    ...aptamers can either fold to incorporate their target molecule, such as a metal ion, or fold to incorporate ...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...highly customizable in terms of how they actually “report” as biosensors. Making aptamers glow The flexibility...increase its fluorescence, essentially mimicking GFP.  Though the aptamer, called Spinach, required longer...Jaffrey lab has since developed new RNA-based scaffolds to contain and stabilize Broccoli cassettes, thereby...

  16. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    Published
    April 25, 2017, 2:30 p.m.
    ...irreversible PA-FP reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating...mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP to display almost... the anionic form, which allows PA-GFP to exhibit up to a 100-fold increase in fluorescence when illuminated...Color Contrast (Fold Change in Fluorescence) Brightness Excitation/ Emission (nm) PA-GFP Monomer UV-Violet...example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) the Youle Lab was able...illuminated with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent PA-FPs by ...in vivo imaging models perfer the PA-RFPs over PA-GFP. Another class of irreversible PA-FPs are the photoconvertible...

  17. Fluorescent Proteins 101: Fluorescent Biosensors

    Type
    Blog Post
    Published
    May 18, 2017, 2:30 p.m.
    ...detects. This change then generates a reporter signal. Reporter signals may be electrochemical or light-based...light-based, with luminescent and fluorescent reporters being especially popular. We’ll give you an introduction...biosensors as an example. Many calcium biosensors include GFP fused to calmodulin (CaM) and the chicken myosin ...calcium-dependent increases in fluorescence of up to 11,000-fold. FRET biosensors are another common class of biosensors...glutamate sensor pCMV(MinDis).iGluSnFR. Picking your reporter Don’t forget about the other end of the biosensor...

  18. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

    Type
    Blog Post
    Published
    Nov. 24, 2015, 3:30 p.m.
    ...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... of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter lines...a UAS-driven luciferase reporter construct. In the absence of GFP, no reporter output was observed in ...electroporated T-DDOGs, GFP, and a reporter construct into the mouse retina. GFP expression robustly activated...T-DDOGs with two mouse GFP reporter lines; again, TdTomato was seen only in cells with GFP, at a high activation...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted...

  19. New Viral Vectors - Fall 2024

    Type
    Blog Post
    Published
    Oct. 3, 2024, 1:15 p.m.
    ...viral prep pAAV-CAG-GFP AAV9-X1.1 Controls Edward Boyden New serotype pAAV-hSyn-EGFP AAV PHP.eB Controls... monkeys it actually targets neurons, and has 45-fold higher expression levels compared to AAV9. You can... can now target these cells with AAV-CAG-GFP packaged in AAV9-X1.1!  We also have eight new AAV-PHP.eB...check out Addgene's Data Hub to see viral vector reports from other users. And don't forget to use the link...Controls Jonathan Ting New viral prep pAAV-CaMKIIa-EGFP AAV1 Controls Bryan Roth New serotype pAAV-Ef1a-DIO...

  20. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ... later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the... obtained using ddPCR are generally two to three fold higher than those achieved using the standard qPCR...expression and/or function. These data are sometimes reported or posted on the material page for the corresponding...viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445-AAVrg) alone at 1.1E6 vg/mL, ...prep # 55632-AAVrg). pAAV-CAG- FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from Edward Boyden (Addgene viral...recipient’s initial thaw will be accounted for in our reported titers. Lentiviral vectors are titered using a...

  21. Visualizing Translation at the Single Molecule Level

    Type
    Blog Post
    Published
    Aug. 1, 2017, 1:15 p.m.
    ...Yan et al.’s reporter contains 24 copies of SunTag, a synthetic scaffold that can recruit GFP fused to the...following components: a GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein...protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein...64543) Before translation, both GFP and RFP are bound to the reporter, causing it to appear yellow. The...translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the reporter. Because it contains...translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known to be important...correlated with GFP intensity, showing that TRICK is an accurate readout of relative reporter translation....

  22. Popular Retroviral Vectors and Their Uses in Scientific Research

    Type
    Blog Post
    Published
    July 24, 2018, 12:51 p.m.
    ...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning and...and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid...a gRNA cassette. It can be used to produce a ~10-fold higher titer of lentiviral particles than the original...together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging studies...

  23. Sleeping Beauty Awakens for Genome Engineering

    Type
    Blog Post
    Published
    June 30, 2015, 2 p.m.
    ...puromycin, hygromycin) and fluorescent protein reporters (GFP, BFP, RFP, no FP). The vectors each contain...SB transposase, SB100X, that is approximately 100-fold more efficient than the first-generation transposase...region of the RPL41 gene, to drive the selection/reporter parts of each construct. This new tool kit allows...

  24. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    Published
    Aug. 19, 2014, 3:39 p.m.
    ...popular studies involve using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following...from the resin and used for various binding or folding assays. Yeast vectors There are several types of...animals, gene targeting and genome modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g... allow you to visualize YGOI in vivo (try pcDNA3-EGFP). If you can get away with generating transiently...

  25. Synthetic Photobiology: Optogenetics for E. coli

    Type
    Blog Post
    Published
    Sept. 8, 2015, 2:30 p.m.
    ...phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly deactivates...highest level of “output” signal, was only about 10-fold, precluding the study of gene expression at very...leakiness and increased dynamic range to around 100-fold. These improvements will allow researchers to better...

  26. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    Published
    Oct. 27, 2015, 3 p.m.
    ...fusion protein molecules fail to correctly fold? Misfolded FPs do not fluoresce. A dark population is ...disrupt FP folding rendering the protein non-fluorescent. Unfortunately, accumulated misfolded and nonfunctional...Lindsey M. Costantini, and Erik L. Snapp. "Superfolder GFP is fluorescent in oxidizing environments when...potentially affect function [5]. We and others have reported that FP cysteine residues form inappropriate disulfide...been a pervasive view that most FPs in a cell will fold and that the fluorescence pattern is equivalent ...brighter FP, but rather because of the correct folding and fluorophore formation by most, if not all, ...missed the impact of dark, non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments...

  27. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    Published
    Oct. 22, 2015, 2:30 p.m.
    ...its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether it interacts...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...genes as reporters and some that repress instead of activate transcription. The HIS3 reporter system (...co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing...referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI...Gal4 activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including...to have Y2H systems that avoid transcriptional reporters altogether; In the split-luciferase system, intein-mediated...

  28. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ...region of a FLAG-binding scFv onto a well-folded scFv scaffold to improve its stability in cells, and use...-FLAG scFv and a stable scFv scaffold. (b) Schematic of anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear...nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets with nuclear (top, 4x-FLAG-mCherry-H2B...induce high levels of gene activation for both reporter and endogenous genes. It is highly specific in...This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and HaloTag fusions,...fusions, while the anti-HA frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG...

  29. Why Add Sucrose? Improved Yields for Adeno-associated Virus Preparation

    Type
    Blog Post
    Published
    Dec. 13, 2018, 1:14 p.m.
    ...following sucrose treatment. Moreover, several reports have shown that cells treated with sucrose or other...of sucrose improve AAV2 yields approximately 1.8-fold without affecting cell viability. This effect appears...endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe any difference...

  30. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...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...gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy...identified a family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on...mutagenesis libraries or to insert heterologous genes, reporters, or other markers into a target genome. They did...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...

  31. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    Published
    Dec. 20, 2022, 2:15 p.m.
    ...stage-specific promoter and design a UAS reporter construct, the reporter will only be expressed where and/or...UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter with...QUAS driven expression by ~3,300-fold in Drosophila cells and ~24,000-fold in human HeLa cells as compared...Church lab uses this system to control expression of GFP.)  To turn this inducible system into a repressible...

  32. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    Published
    April 19, 2016, 2:30 p.m.
    ...Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald, CFP, YFP, BFP, Cerulean...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...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...

  33. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    Published
    Jan. 29, 2019, 2:27 p.m.
    ...poor folding and thermal instability at 37°C (Ouellet et al., 2016) and requires a tRNA scaffold to improve...aptamers able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...Broccoli increases its fluorescence by about 1.8-fold, and may be a better choice for imaging than the...complex and the entire aptamer can hybridize and fold. (Alam et al., 2017). Yellow fluorescent aptamers...growth as RNA biology, we are looking forward to reports on the next additions to aptamer soup! References...that was more thermally stable, exhibited less misfolding, and was consequently brighter than Spinach. ... Matthew D. Disney, and Samie R. Jaffrey. "A superfolding Spinach2 reveals the dynamic nature of trinucleotide...

  34. Hot Plasmids: Summer 2024

    Type
    Blog Post
    Published
    Aug. 27, 2024, 1:15 p.m.
    ...capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph of H1.8-GFP nucleosomes collected...In MagIC-cryo-EM, target proteins (expressed as a GFP fusion in this proof-of-concept study) are captured...SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines ...) 3D model of the obtained structure of the H1.8-GFP-bound nucleosome. Images adapted from Arimura et ...Integrase Gene Editing (PASSIGE), their previously-reported technique for precisely integrating large DNA ... integration efficiency of 23%, a more than four-fold improvement over the wild-type enzyme. Find optimized...

  35. Split Fluorescent Proteins for Studying Protein-Protein Interactions

    Type
    Blog Post
    Published
    May 3, 2022, 1:15 p.m.
    ...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... (abundant in eukaryotes) A Near-Infrared BiFC Reporter for In Vivo Imaging of Protein-Protein Interactions...green-yellow or orange-red fluorogen A split fluorescent reporter with rapid and reversible complementation. Tebo...

  36. Hot Plasmids: Fall 2024

    Type
    Blog Post
    Published
    Nov. 21, 2024, 1:30 p.m.
    ...gene disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag antibody ...Western Blot for Anti-V5 [IPI-SV5-Pk1]. Addgene Report, https://doi.org/10.57733/addgene.l0ulu4. Hanke...considerably faster, with expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3...through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3 and...doi.org/10.1128/jb.00335-23.  Bioluminescent reporting of mechanical forces with PILATeS By Emily P. ... reaction (Figure 5). The researchers used the reporter to visualize biologically relevant forces at focal... focal adhesion marker paxillin (visualized with GFP). Reprinted with permission from Zhong et al, 2024...

  37. Hot Plasmids September 2018 - Optogenetics, RNA Localization, Fluorescent Protein, and Base-editing Tools

    Type
    Blog Post
    Published
    Sept. 18, 2018, 1:33 p.m.
    ...ACE) in real-time. The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry...maximum is reported as 603 nm, while the emission maximum is 675 nm. mCarmine is monomeric, 4-5 fold brighter... ACE system: A versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9...editing tools, the Reuben Harris lab has deposited a reporter plasmid that allows for rapid fluorescence-based... out a frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply...containing rat APOBEC1. Finally, they show that the ACE reporter can also quantify Cas9 cleavage, thus exhibiting...editing activities. Click here to find the ACE reporter plasmid! Martin et al., Nucleic Acids Research...

  38. Quick Guide to Near-Infrared Fluorescent Proteins

    Type
    Blog Post
    Published
    Oct. 5, 2021, 1:15 p.m.
    ...molecular brightness (Table 1).  NIR reporters and biosensors NIR reporters can tell us about cellular events...normalization to fluorescence of co-transfected EGFP. e Originally reported as a monomer16, IFP2.0 was later found... state-of-the-art NIR FPs can be used similar to GFP-like probes by supplying a single gene to cells. ...crosstalk-free three-color imaging with green and red GFP-like FPs (Shcherbakova et al., 2016), as well as ...higher apparent cellular brightness than previously reported widely used dimeric iRFPs (Table 1). In general...protein-protein interactions, we recommend using NIR split reporters based on miRFPs (Shcherbakova et al., 2016), since...E., Sivanesan, D. & Michnick, S.W. An infrared reporter to detect spatiotemporal dynamics of protein-protein...

  39. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...phosphatase-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked mCherry...elements and combined it with a variety of reporters (i.e. eGFP) and effectors (i.e. DREADDs). The team ...are available at Addgene. Kim, et al. Stem Cell Reports. 2015. PubMed PMID: 25772473 Targeting and manipulating...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...off-target cutting, and they found that HF-BE3 showed 37-fold less off-target editing than BE3, with only a slight...reagents shared via Addgene. Narayan S, et al. Cell Reports. 2017. PubMed PMID: 28813671 Listen to the podcast...along with EGFP while retaining an emission spectrum that is easily separable from that of EGFP. This makes...

  40. Fluorescent Protein Travel Awards - Protein Variants, a Serotonin Sensor, and an Artificial Leaf Replica System

    Type
    Blog Post
    Published
    June 4, 2019, 1:02 p.m.
    ..., Bernach plans to use a suite of fluorescent reporters. Bacteria containing unstable fluorescent proteins...colonizer, labelled with a constitutively fluorescent reporter can also be used to track how one population responds...using TIMER and an unstable fluorescent protein reporter under the control of promoters that control cell...fluorescent protein topics page Get the basics on GFP Find microbiology blog posts Resources on Addgene.org... the TIMER growth rate bioreporter. During protein maturation, the bioreporter spontaneously changes from...decreased in protein abundance, possibly due to misfolding. But measuring variants one at a time is time...variant library is monitored by fusing the variants to eGFP followed by an internal ribosomal entry site and...

  41. A Practical Approach to Choosing the B(right)est Fluorescent Protein

    Type
    Blog Post
    Published
    June 1, 2017, 2:30 p.m.
    ...of posts explains how to critically assess the reported properties of fluorescent proteins, how to do ...protein with a high intrinsic brightness that does not fold well or cannot be detected in a microscopy setup...experimental conditions into account. In particular, the folding and maturation efficiency of the fluorescent protein... QY (EC*QY, sometimes normalized to the value of EGFP). The higher the number, the higher the theoretical...

  42. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

    Type
    Blog Post
    Published
    June 17, 2021, 1:17 p.m.
    ... strain transformed with pBLADE expressing superfolder GFP instead of mCherry under the PBAD promoter....the DNA-binding domain of AraC using a mCherry reporter downstream of a PBAD promoter. While none of the...contains BLADE, the PBAD promoter, and a mCherry reporter downstream of the PBAD promoter. To use this plasmid...

  43. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ..., Hess et al. successfully evolved wild type GFP to EGFP using CRISPR-X and subsequent FACS sorting. They...-ome using a quick, affordable, and ubiquitous reporter assay. In their recent publication, Kroeze et ...with unique tissue-specific promoters expressing GFP, tetracycline response elements, and shRNAs many ... different human cell models and identified ~4-5-fold more fitness-related genes than have been found ... of DsRed connected to a pH-sensitive variant of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The...when it is difficult for a cell to synthesize or fold a protein because of its length. Mark Howarth’s ...endogenous proteins. As described in their recent Cell Reports paper, the Kanemaki lab overcame this hurdle using...

  44. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    Published
    Sept. 21, 2017, 2:07 p.m.
    ... using a superfolder GFP fusion to stabilize the protein in mammalian cells. LwaCas13a-msfGFP can mediate...of interest and an inactivated fluorescent RNA reporter. If the target sequence is present in the pool...activity of Cas13a becomes activated and the RNA reporter will be cleaved resulting in activation of the...

  45. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...destination vector contains GFP, enabling you to select cells with high GFP expression. These cells have...plasmids, which contain various promoters and reporters, and subsequently inserted into a Cas9-containing...-in-one CRISPR/Cas9 vector system.” Scientific Reports 4 (2014): 5400. PubMed PMID: 24954249. PubMed Central...vectors containing the U6 promoter and the gRNA scaffold are provided with the kit. Oligonucleotides specifying...

  46. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    Published
    Oct. 12, 2023, 1:15 p.m.
    ....1 showed the greatest fold change in RNA and DNA levels (~25-fold and ~4-fold respectively) relative ...expression from mouse to primate neocortex. Cell Reports, 34(13). https://doi.org/10.1016/j.celrep.2021.108754...relatively even distribution of target gene expression (EGFP, in the depositing manuscript) across the brain....
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