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We narrowed to 65 results for: IRES-GFP

Showing: 1 - 50 of 65 results

  1. New and Upcoming Viral Vectors - May 2020

    Type
    Blog Post
    Published
    May 28, 2020, 1:15 p.m.
    ...2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro:...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro) are also in the...respectively of a GFP dependent Cre. The two segments come together in the presence of GFP to form a functional... activation to cells expressing both Cre and GFP. The GFP dependent FLPo, Flp-DOG (75469), is also available...-2 proteins ChR2 for targeted expression DREADDs GFP dependent Cre and FLPo And more!  New lentiviral...M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a Lentiviral...in new serotypes, as well as a new Cre dependent GFP control plasmid for expression in forebrain GABA-...

  2. New and Upcoming Viral Vectors - Spring 2019

    Type
    Blog Post
    Published
    March 26, 2019, 1:40 p.m.
    ...GlyR IRES EGFP Chimeric channels for neuronal manipulation 119742  AAV5  AAV SYN PSAM4 GlyR IRES EGFP...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...37825  AAV8*, AAV9*  pAAV-CAG-GFP 50465  AAV8*, AAV9*  pAAV-hSyn-EGFP *Also coming in our new 20 ...pAAV-GFAP104-mCherry, pAAV-mDlx-NLS-mRuby2, CAG-NLS-GFP,and pAAV-hSyn-mCherry at Addgene. Viral vectors coming...-dependent CRE) 55634  AAV1  pAAV-EF1a-mCherry-IRES-Flpo 55637  AAV1  pAAV-EF1a-Flpo Calcium sensors...

  3. New and Upcoming Viral Vectors - June 2019

    Type
    Blog Post
    Published
    June 18, 2019, 1:15 p.m.
    ...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... Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP Recombinase AAV Finally, we are excited to ...testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP Recombinases...Serotype Name 119472  AAV5  AV SYN PSAM4 GlyR IRES EGFP   Biosensors Plasmid Serotype Name 105322...various serotypes of two popular control AAV encoding GFP. Check out our inventory of serotype testing AAV ...)-mCherry 50467  AAV2 pAAV-CaMKIIa-HA-hM4D(Gi)-IRES-mCitrine 121538  AAV5 pOTTC1484 - pAAV SYN1 HA-hM4D...

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

    Type
    Blog Post
    Published
    May 15, 2014, 3:33 p.m.
    ...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...

  5. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ...pAAV-Ef1a-mCherry-IRES-Cre (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2]... later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the...expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre was a gift from Karl Deisseroth (Addgene viral...prep # 55632-AAVrg). pAAV-CAG- FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from Edward Boyden (Addgene viral...

  6. 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...

  7. Tagging Optogenetics and Chemogenetics Receptors: Fluorescent Proteins and Other Options

    Type
    Blog Post
    Published
    May 11, 2021, 1:15 p.m.
    ...protein of interest. The green fluorescent protein (GFP) and mCherry, two of the most common tags currently...with the opsin or chemogenetic receptor, through an IRES or 2A sequence. In this case, the proteins are not...article on the Addgene blog: Fluorescent Proteins 101: GFP Fusion Proteins – Making the Right Connection.   ...

  8. 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 ...

  9. Visualizing Translation at the Single Molecule Level

    Type
    Blog Post
    Published
    Aug. 1, 2017, 1:15 p.m.
    ...enable this color switching, TRICK requires the following components: a GFP protein that binds to the coding... a 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...

  10. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    Published
    March 21, 2017, 2:30 p.m.
    ...containing the gene of interest (pTet-IRES-EGFP, pPRIME-Tet-GFP-FF3) and one with either tTA or rtTA (...be separated by an internal ribosome entry site (IRES) and expressed under the same promoter. Alternatively...viral titre to the number of cells transduced by a GFP encoded virus, and the reverse transcriptase activity...in terms of RCL generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for...that there is often  background activity and it requires continuous administration of Dox to repress transgene...One drawback of the Tet system is that it often requires the delivery of two distinct expression vectors...

  11. 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...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...than HRP-RANbody staining Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B Myc Tagged RFP...sensitive than HRP-RANbody staining Requires secondary antibody staining GFP, RFP, H2A2B   Are you ready... 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...

  12. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    Published
    July 7, 2020, 1:15 p.m.
    ...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...

  13. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ... 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 ...

  14. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    Published
    May 20, 2014, 2:06 p.m.
    ... 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...on many FPs (particularly those derived from GFP) requires oxygen. Therefore, these FPs cannot be used ...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...

  15. 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...background signal (especially for some older split-GFP constructs or certain cell environments). Also consider... variant of mScarlet Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of...

  16. New and Upcoming Viral Vectors - September 2019

    Type
    Blog Post
    Published
    Sept. 12, 2019, 1 p.m.
    ...collection is now complete! pAAV-CAG-GFP (plasmid 37825) and pAAV-hSyn-EGFP (plasmid 50465) are now available...DreO-bGHpA (50363-AAV5) Flp vectors pAAV-EF1a-mCherry-IRES-Flpo (55634-AAV1) pAAV-EF1a-Flpo (55637-AAV1) Viral...our entire AAV inventory. Our new AAVs include: EGFP-expressing AAV for serotype testing Calcium sensors... testing AAV, which are small (20 ul) samples of EGFP-expressing AAV packaged in various serotypes. This...recently released these controls: pAAV-hSyn-DIO-EGFP (50457-AAV5): The Synapsin promoter directs broad...broad, neuronal expression. AAV pCAG-FLEX-EGFP-WPRE (51502-AAV5): The CAG promoter directs ubiquitous expression...interneurons. pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP (112677-AAV5 and 112677-AAVrg): The EF1a promoter...

  17. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    Published
    Jan. 24, 2023, 2:15 p.m.
    ...P2A-linked accessory genes gvpNJKFGWV marked with Emerald GFP. These plasmids can be used directly in transient...gating for the top quartile (or higher) of BFP and GFP producing cells. After FACS, return the cells to ...Simply cotransfect a mixture of 1248 ng of CMV-gvpA-IRES-BFP plasmid and 954 ng CMV-gvpNJKFGWV plasmid into...

  18. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    Published
    April 25, 2017, 2:30 p.m.
    ...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...

  19. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    Published
    Dec. 1, 2016, 3:30 p.m.
    ...recombinase. If GFP is under control of an endogenous promoter, you can use expression GFP to track cells...tag an endogenous protein with GFP, as seen in blue flame plasmid OCT4-eGFP-PGK-Puro from the Jaenisch lab...In order to both knockout a given gene and insert GFP into the genome, we’d create a plasmid similar to...to the one shown below, where the sequence of GFP is cloned together with the Neomycin resistance (NeoR... 3 of the targeted gene. Upon recombination, the GFP/NeoR cassette is inserted in place of exon 2. Thus...gene is disrupted (knocked out) but the inserted GFP is expressed (knocked in). As seen in the above example...knockout/knock-in targeting vector designed to insert GFP into a given locus.   Conditional knockouts ...

  20. Scientists Map the SARS-CoV-2-Human Interaction Network

    Type
    Blog Post
    Published
    April 9, 2020, 1:15 p.m.
    ...the viral protease, Nsp5. Our set also includes a GFP construct as a control. For easier portability, all...vectors if you need to. Additionally, they have an IRES-Puro marker to facilitate stable cell clone selection...

  21. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    Published
    May 28, 2024, 1:45 p.m.
    ...overlap. Figure 4 compares the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore...to fuse them with a fluorescent reporter, such as GFP or mCherry, through genetic modification. As your... you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies together in...while wavelengths over 700 nm make up the infrared (IR) spectrum. When photons are absorbed by matter, they...vector also encodes for eGFP, which serves as a transduction reporter. Note that eGFP is not fused to CD45...-Cy7. Next comes our transduction reporter, eGFP. As eGFP emits fluorescence by itself and does not need...cytometer which channel to use to detect eGFP. As you saw in Figure 4, eGFP strongly overlaps with FITC, hence...

  22. 15 Years of Addgene: The Top 15 Plasmids

    Type
    Blog Post
    Published
    Jan. 8, 2019, 1:54 p.m.
    ...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...lab that contains the gag and pol genes. It also requires pRSV-Rev and the envelope expressing plasmid pMD2...

  23. 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...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.,...

  24. Multiplex Genome Editing with CRISPR-Cpf1

    Type
    Blog Post
    Published
    May 9, 2017, 2:12 p.m.
    ...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...transduction One vector expresses Cas9, eGFP, and up to 4 gRNAs Each gRNA requires its own promoter   Kabadi et ...crRNA/gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5'...vector expresses Cas9 and up to 7 gRNAs Each gRNA requires its own promoter Sakuma et al   Multiplexed...gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9 Nissim et alTsai et al...

  25. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    Published
    Feb. 28, 2017, 3:30 p.m.
    ...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...of these orthologs is key: since each ortholog requires a different PAM sequence, a gRNA designed for ...color is specified by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence...in combination with gene editing. CRISPRainbow requires very short, 11-mer gRNAs, which do not induce ...

  26. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    Published
    June 23, 2016, 2:30 p.m.
    ...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 #... The generation of pSer-incorporated proteins requires a molecular toolkit containing the OTS, an appropriate...

  27. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    Published
    Sept. 9, 2014, 8:20 p.m.
    ...MSCV-IRES-EGFP IRES Retroviral pMSCV-pBabeMCS-IRES-RFP IRES Retroviral pMSCV-IRES-YFP II IRES Retroviral...pCMMP-MCS-IRES-Puro IRES Retroviral pEF1a-IRES-Neo IRES Mammalian MSCV-IRES-Luciferase IRES Retroviral...plasmids for research purposes are described below. IRES Elements Translation in eukaryotes usually begins... element called an Internal Ribosome Entry Site (IRES) to allow for initiation of translation from an ...mRNA. In the figure above, you can see that the IRES element acts as another ribosome recruitment site...co-expression of two proteins from a single mRNA. IRES was originally discovered in poliovirus RNA, where...in eukaryotic cells.1,2 Since then, a variety of IRES sequences have been discovered - many from viruses...

  28. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    Published
    Aug. 15, 2017, 1:24 p.m.
    ... 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...Laser include: Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, ...auto-fluorescence (Drobizhev et al. 2011). But this requires other lasers than the standard Ti:Sapphire laser...

  29. Choosing Your Fluorescent Proteins for Multi-Color Imaging

    Type
    Blog Post
    Published
    Oct. 9, 2014, 3 p.m.
    ...imaging with this set are mTagBFP2, EGFP or one of the improved GFP variants, mRuby2 or TagRFP-T, and an... / em 527 nm) can be imaged together but CFP and GFP (ex 488 nm / em 507 nm) show some crosstalk between...simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation... means that the choice of fluorescent proteins requires some thought. The first step in choosing fluorescent...fluorescent proteins can be separated from each other requires knowledge of their excitation and emission spectra...mammalian cells, one of the improved folding variants of EGFP like mEmerald or Clover is probably best; mNeonGreen...and have reported brightness measurements. Here, EGFP outperforms the improved folding variants, presumably...

  30. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...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     ...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...monoclonal antibodies against them, fused them to GFP, and compared them to popularly used epitopes tags...

  31. Visualizing Genomic Loci with CRISPR-Sirius

    Type
    Blog Post
    Published
    April 18, 2019, 12:53 p.m.
    ...each recruits a different fluorescent protein (BFP, GFP, or RFP) to the genomic loci of interest. Pairs of...hairpin sequence (sgRNA-Sirius-8XPP7) to recruit GFP to the loci of interest. To determine how the sgRNA-Sirius...different chromosomes in living cells, the system requires high-copy chromosome-specific loci in the genome...

  32. Antibodies 101: Single Chain Fragment Variables (scFvs)

    Type
    Blog Post
    Published
    June 3, 2021, 1:15 p.m.
    ...binding scFv that’s fused to GFP. When the multiple copies of the scFv-GFP fusion bind the SunTag scaffold...scFv that’s fused to a fluorescent protein such as GFP. The HA frankenbody works just like an antibody-based...for the scFv (Gaciarz and Ruddock., 2017), but requires an oxidizing environment to form. When expressed...

  33. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ...anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling...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...label your favorite protein! While the target only requires a small epitope tag (FLAG is eight amino acids...

  35. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

    Type
    Blog Post
    Published
    Sept. 26, 2019, 12:50 p.m.
    ...hiPSCs  EMX1  67% Mouse BM cells from GFP transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~...with CRISPR components. However, this sometimes requires intracellular injections into the cytoplasm or...

  36. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    Published
    Dec. 17, 2020, 1:30 p.m.
    ...drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes...homology arms to knock in large epitopes, such as GFP (Paix et al., 2014). A further development of this...efficiencies, obtaining as many as 100 independent GFP knock-ins from a single injected animal.  PAM sequence...sequence added, leaving a 34 bp “scar”, but currently requires additional experimental manipulation. In some ...

  37. Better Dyeing Through Chemistry & Small Molecule Fluorophores

    Type
    Blog Post
    Published
    Sept. 8, 2016, 2:30 p.m.
    ...chemistry! The discovery of green fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly...advances lead to an obvious question: In this age of GFP and its ilk, why should cell biologists talk to chemists...the frontiers of fluorescence microscopy often requires more photons. For example, moving from transient...

  38. Plasmids 101: Protein tags

    Type
    Blog Post
    Published
    Dec. 11, 2014, 4:26 p.m.
    ...protease protection. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes... I Use? Plasmids 101: Green Fluorescent Protein (GFP) Fluorescent Protein Guide Expression Vectors from...cytometry, but rarely used for purification as elution requires low pH poly His HHHHHH  0.8 Affinity and Purification...

  39. Antibodies 101: Affinity Tags

    Type
    Blog Post
    Published
    Sept. 19, 2023, 1 p.m.
    ... (FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry, etc.). Fluorescent tags are primarily used...must be performed after primary purification and requires a second clean up step to remove the cleaved tag...Also, purifying GST by affinity chromatography requires proper folding of the tag, which prevents insoluble.... Mammalian purification is still possible but requires optimization and often more stringent washing....

  40. New Viral Vectors - March 2024

    Type
    Blog Post
    Published
    March 5, 2024, 2:15 p.m.
    ...Biosensor Looger New serotype pAAV-CAG-DIO-NLS-mRuby3-IRES-eGtACR1-ST AAV9 Optogenetics Adesnik New viral vector...vector pENN.AAV.EF1a.eGFP.WPRE.rBG AAV PHP.eB Controls Wilson New serotype pAAV-CaMKIIa-EGFP AAV PHP.eB...Chemogenetics Deisseroth New viral vector pAAV.CMV.PI.EGFP.WPRE.bGH  AAV PHP.eB Controls Wilson New viral ...

  41. New Norepinephrine Indicators: nLightG and nLightR

    Type
    Blog Post
    Published
    Dec. 12, 2023, 2:15 p.m.
    ...contains circularly permuted green fluorescent protein (GFP), and nLightR uses a red fluorescent protein, mApple..., Wu, Z., Wu, H., Chen, W., Zhang, P., Zou, J., Hires, S. A., Zhu, J. J., Cui, G., Lin, D., Du, J., & ...

  42. Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation

    Type
    Blog Post
    Published
    May 21, 2020, 1:15 p.m.
    ...transcription activation systems, such as LexA/p65 or mCD8/GFP, to allow sparse labeling in a variety of fly cell...transgene (Lin et al., 2018). However, this system requires precise titration of the AAV. So far, tools to...

  44. Viral Vectors 101: Parts of the AAV Transfer Plasmid

    Type
    Blog Post
    Published
    Aug. 11, 2020, 1:15 p.m.
    ...or smaller. Some examples include a reporter like GFP, some smaller CRISPR-Cas proteins such as SaCas9,...this awesome experiment you want to do, but it requires some AAV. You’ve never worked with AAV before,...

  45. Using Addgene's New Viral Service for Your Research

    Type
    Blog Post
    Published
    Nov. 17, 2016, 3:30 p.m.
    ...also have lentivirus for delivering control shRNAs, GFP, and rtTA from the Tet-on system. The first two can...Virus production can be a frustrating process that requires a large investment of time and resources for labs...

  47. 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...editing, and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed...

  48. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    Published
    Aug. 10, 2023, 1 p.m.
    ...ones? Many of the available biosensors use a single GFP or CFP/YFP-derived FRET pair. If you need to free...molecular neuroscience. 2014 Nov 18;7:88. Tian L, Hires SA, Looger LL. Imaging neuronal activity with genetically...

  49. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    Published
    April 3, 2014, 8:05 p.m.
    ...Vectors Read about Reporter Gene like Luciferase and GFP Resources on Addgene.org Find Plasmids for Your...binding sites, commonly used in Drosophila Specific Requires the presence of Gal4 gene to activate promoter...Promoter from T7 bacteriophage Constitutive, but requires T7 RNA polymerase. When used for in vitro transcription... Negligible basal expression when not induced. Requires T7 RNA polymerase, which is also controlled by...Promoter from Sp6 bacteriophage Constitutive, but requires SP6 RNA polymerase. SP6 polymerase has a high ...Promoter from T3 bacteriophage Constitutive, but requires T3 RNA polymerase When used for in vitro transcription...

  50. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

    Type
    Blog Post
    Published
    Aug. 27, 2020, 1:15 p.m.
    ...check out our article: Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection. Cellular... of hard-to-achieve factors. For example, FRET requires that fusion proteins be in close proximity and...FRET, as the overexpression of fusion proteins it requires can lead to aberrant subcellular localization ... construction Fusing FP fragments to your PIPs requires careful selection of FP fragments and, preferably...information, constructing appropriate controls may requires that you empirically test a number of mutants ...
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