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Showing: 1 - 20 of 152 results
  1. Seeing Red: Simple GFP Photoconversion

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    Nov. 20, 2019, 4:33 p.m.
    ...gfp...This imaging process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red...After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP...dynamics can be undertaken using available GFP lines....Tobacco suspension cell culture with cytosol-targeted GFP....GFP is the expression level necessary to observe a signal....Green to red photoconversion of GFP for protein tracking in vivo....Photoconverted GFP is compatible with other FPs since its emission wavelength (612 nm) is distinct from...not be appropriate for high-pH cellular compartments....FPs like PsMOrange and mEos add another layer of complexity to imaging, as they allow one to monitor compartment...This imaging process worked well with the common GFP variants EGFP, mGFP4-T, and S65T GFP, and no red...
  2. Visualizing Translation at the Single Molecule Level

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    Oct. 13, 2019, 9:56 p.m.
    ...To enable this color switching, TRICK requires the following components: a GFP protein that binds to...the coding region of a reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP protein that binds...The act of translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the reporter...Oskar protein levels were negatively correlated with GFP intensity, showing that TRICK is an accurate...proteins and is driven by an inducible promoter (Plasmid 64542 or 64543) Before translation, both GFP...cycloheximide and puromycin, Halstead et al. verified that translation is necessary for removal of GFP...Additional Resources the Addgene Blog Learn More about SunTag Catch up on Your GFP Background Choose...Before beginning experiments, it’s important to verify that these changes do not impact translation rate...mRNAs to the plasma membrane, it’s not appropriate for use with mRNAs usually translated in a specific compartment...
  3. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

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    July 7, 2020, 1:15 p.m.
    ...As a proof of concept the Dow lab tested their hypothesis with GFP fluorescence....TLDR: TREE from the Wang and Brafman lab uses an engineered BFP variant that converts to GFP after a...GFP positive cells can be sorted and have a significantly higher frequency of base edits of interest...As expected, only the cells that contained the cytidine base editor exhibited robust induction of GFP...The Wang and Brafman lab showed that GFP positive cells isolated with TREE have a significantly higher...Nature 551:464–471 . https://doi.org/10.1038/nature24644 Glaser A, McColl B, Vadolas J (2016) GFP to...Thus a successful base editing event would result in GFP fluorescence that can be visualized and sorted...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...frequency of genomic base pair edits of interest compared to cells segregated solely by a reporter of...using Fluorescence-activated cell sorting (FACS) and the editing efficiency can be easily determined by comparing...This created three inactivated eGFP reporters- eGFP L202, eGFP L138, and eGFP L93....cells expressing only eGFP expression to cells expressing eGFP and mCherry....The eGFP reporters rely on the correction of point mutations in eGFP that results in the restoration...This edit changes the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission...
  4. Plasmids 101: SunTag and Fluorescent Imaging

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    Oct. 20, 2019, 9:08 p.m.
    ...gfp...Initially, Tanenbaum et al. observed some GFP aggregation, which they reduced by using superfolder GFP...the effects of mitoNEET-SunTag-GFP....has a molecular weight of 1,400 kDa vs 24 kDa for GFP alone....In SunTag nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.  ...GCN4 recruits GFP fused to the cognate scFV antibody, which is expressed from a separate plasmid....Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they examined...Having seen that mitochondrial tracker GFP-mitoNEET can impair mitochondrial function, they examined...Pros of SunTag compared to traditional fluorescent proteins Improved brightness and signal-to-noise...In SunTag nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.  ...(sfGFP) with the small solubility tag GB1....
  5. PITChing MMEJ as an Alternative Route for Gene Editing

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    Nov. 19, 2019, 3:54 p.m.
    ...One potential concern is if the GFP-Puro will alter expression of the gene it follows....They tested their system in HEK293 cells, integrating the GFP-Puro cassette into the FBL locus....Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without...Three double stranded breaks are necessary for knock-in: one on either side of the GFP-Puro cassette...alternative systems The ready-made PITCh plasmids available from Addgene are perfect for expressing GFP...the lab’s previous work, Sakuma et al. describe a detailed protocol for MMEJ-mediated knock-in of a GFP-Puro...PITCh vector should be constructed with 5’ and 3’ microhomology to the insertion locus flanking the GFP-Puro...Compared to its counterparts NHEJ and HDR, MMEJ doesn’t get a lot of press....One important question to ask is how the efficiency of PITCh-based genomic insertion would compare to...
  6. RANbodies: Reporter Nanobody Fusions

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    Dec. 14, 2019, 2:08 a.m.
    ...Some GFP nanobodies  stain poorly when tagged with a Spaghetti Monster because they recognize this GFP...GFP, RFP) Detecting GFP reconstitution across synaptic partners (GRASP) assays No antibody required..., Yamagata et al used previously published nanobody sequences that targeted the following antigens: GFP...can be cumbersome Tyramide reagents can be expensive Can only stain with one HRP-RANbody at a time GFP...secondary Less sensitive than HRP-RANbody staining Requires secondary antibody staining HA Tagged GFP...labeled secondary   Less sensitive than HRP-RANbody staining Requires secondary antibody staining GFP...Spaghetti Monster Spaghetti Monsters are highly antigenic tags that have 10 HA or 10 MYC tags built into a GFP...GFP....Compatible with multi-labeling protocols when other antigens are detected with either a labeled primary...HRP-RANbodies are, however, compatible with most multi-labeling immunohistochemical protocols where either...likely due to lower background and better penetration of the sample due to the smaller size of RANbodies compared...
  7. Which Fluorescent Protein Should I Use?

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    Oct. 18, 2019, 12:17 a.m.
    ...week on GFP....GFP also emits yellow light)....GFP, Venus) can be split into two halves, which are non-fluorescent on their own....By mutating GFP, the variants blue FP (BFP), cyan FP (CFP), and yellow FP (YFP) were derived....(FPs) were first discovered over 50 years ago, with the discovery of the Green Fluorescent Protein (GFP...For a breakdown of GFP, it's variants, and their relevant mutations, check out Marcy's post from last...Oxygen: The maturation of the chromophore on many FPs (particularly those derived from GFP) requires...In many cases the brightness is compared to that of EGFP which is set as 1....For example, superfolder GFP (sfGFP) and mNeonGFP can fold in <10min at 37°C, mCherry takes ~15min, TagRFP...For instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most suited for mammalian...Some proteins are reversibly switchable (e.g. rsEGFP, Dreiklang)....
  8. FlipGFP, a novel fluorescence protease reporter to study apoptosis

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    Jan. 3, 2020, 7:44 p.m.
    ...β-strand of GFP, hence the name FlipGFP...."A new protein-protein interaction sensor based on tripartite split-GFP association." ..."Rational design of a GFP-based fluorogenic caspase reporter for imaging apoptosis in vivo."...GFP contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (...from UCSF designed an improved apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP...GFP fluorescence increased over time and showed a clear pattern in the developing forebrain and retina...To do this they modified a different self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single...FlipGFP increases fluorescence 100-fold compared to other reporters (Zhang et al., 2019)....β-strand of GFP, hence the name FlipGFP....split GFP (GFP1-10 and GFP11) to increase fluorescence 10-fold after protease activity (To et al., 2016...To do this they modified a different self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single...How does FlipGFP work?...Studying apoptosis in vivo using the FlipGFP-based caspase reporter The Shu Lab successfully used FlipGFP...You can find the plasmids for FlipGFP and FlipCherry at Addgene....
  9. Neuronal labeling with Spaghetti Monster

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    Jan. 7, 2020, 6:34 p.m.
    ...GFP emits light in only a single channel....The issue with these FPs is that they are less photostable than GFP....GFP and other FPs have the tendency to brightly label somata but not neuronal processes....Multi-channel connectomic tracers Fluorescent proteins such as GFP have been used for a long time as...Visualization of neuronal sub-cellular structures GFP is limited in its ability to label subcellular...in the stratum oriens and the stratum lacunosum moleculare were better resolved with smFP_FLAG than GFP...retain their ability to be detected after sample preparation better than standard proteins tags like GFP...As compared to epitopes tags, FPs are intrinsically fluorescent and can therefore be used in live imaging...For instance, smFPs have been used to visualize “thorny excrescence” (TE) spines in the Hippocampal CA3..., the church of the Flying Spaghetti Monster; the smFP structure resembles images of the deity (see comparison...
  10. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

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    Oct. 27, 2019, 3:13 p.m.
    ...A virus encoding GFP plus a gRNA targeting GFP (pXPR_011, 59702-LV) was used to assay Cas9 activity in...I thought I could see GFP expression with a fluorescence microscope, but the GFP expression was so low...out the virally-delivered GFP gene....You could probably also do Western blotting against GFP expression, but I didn’t. 2. ...In the original publication they see GFP expression in about 20% of their Cas9-expressing cells....(even in the transduced wild-type A549 cells that lack Cas9 and thus should have expressed GFP) that...Using this virus, it’s expected that Cas9 activity, guided by the virally-delivered GFP gRNA, will edit...The digestion product (Figure 4, lanes 0.3 % 3.0, Figure 5, lane 2) is faint, so comparing it to the...I compared two MOIs of gRNA transduction to see if that had any effect on the editing efficiency, and...For comparison, in 2007 it took about 3 months to knockout a single allele in a human line, using methods...with multiple gRNAs, but the most important is that you could avoid potential off target artifacts by comparing...
  11. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

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    May 26, 2020, 1:15 p.m.
    ...The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity. ...If there’s GFP expression, presumably the endogenous target site will be cut and repaired by NHEJ as...genome edit:  A donor plasmid that contains a puromycin resistance gene linked to an in-frame H2B-GFP...target site (the same sequence you’d like to mutate in the genome) and an out-of-frame H2B (histone)-GFP...Lastly, once a mutant is identified, the PB transposase can easily and seamlessly remove the integrated GFP...reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied donor plasmid will express GFP...In an experiment that targeted the gene NFE2L2, the Madison lab found that H2B-GFP expressing cells exhibited...a 2-4 fold increase in the number of endogenous indel edits when compared to unsorted cells (Wen et...As a proof of concept the Denham lab compared editing at three different genomic sites in the AAVS1 locus...By using the RFP:GFP ratio, edited cells can be sorted from unedited cells....
  12. AAVs CREATed for Gene Delivery to the CNS and PNS

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    Nov. 24, 2019, 5:09 p.m.
    ...To quantify this comparison, capsids were packed with a GFP transgene and GFP intensity and transduction...A GFP transgene was packaged into either an AAV.9 or an AAV.PHP.S capsid....A GFP transgene was packaged into either an AAV-PHP.B or an AAV.PHP.eB capsid....On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains...They also transduced a higher number of neurons overall (GFP+ cells that also stained for a neuronal...viruses were IV injected at a dose of 1x1011 vg per mouse and the level of transduction, measured via GFP...viruses were IV injected at a dose of 1x1012 vg per mouse and the level of transduction, measured via GFP...To quantify this comparison, capsids were packed with a GFP transgene and GFP intensity and transduction...On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains...Figure 3 shows a gross comparison of AAV.PHP.eB and AAV.PHP.S to their parental vectors....
  13. Synthetic promoter AAVs for cell-type specific expression in retinal cells

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    March 12, 2020, 1:19 p.m.
    ...marker (CatCH-GFP)....Expression of GFP will only result in cells where Cre-mCherry is also expressed....expression of the same transgene: an optogenetic reporter, channelrhodopsin, fused to a fluorescent GFP...promoter AAVs were first injected into the eyes of mice to test for cell type-specific expression of GFP...and ganglion cells, only the horizontal cells transduced by both AAVs would express Cre-mCherry and GFP...expression of Cre-mCherry and a second promoter to drive expression of a Cre-dependent fluorescent CatCH-GFP...If Cre-mCherry expresses in photoreceptors and horizontal cells, and Cre-dependent-GFP expresses in horizontal...By comparing the performance of promoters across these three species, the Roska lab concluded that a...Comparing synthetic promoters performance in mouse, monkey, and human retinas Next, the 113 AAV promoters...preps Browse all viral vectors References Juettner, J., Szabó, A., Gross-Scherf, B., Morikawa, R.K., Rompani...targeted expression in specific cell types (ex: rods or cones) or cell classes (ex: photoreceptors which encompassed...
  14. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

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    Oct. 20, 2019, 8:15 p.m.
    ...The T203H mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP...PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating Threonine 203 to Histidine...The first successful irreversible PA-FP reported was PA-GFP...."A photoactivatable GFP for selective photolabeling of proteins and cells."...Similar to PA-GFP, scientists later developed red fluorescent PA-FPs by performing several rounds of...irreversible photoconversion of the chromophore from the neutral to the anionic form, which allows PA-GFP...For example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) the Youle Lab...Another important parameter to consider is the contrast ratio- a comparison of PA-FP brightness before...PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating Threonine 203 to Histidine...For example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) the Youle Lab...When wtGFP is illuminated with UV-violet light (~400 nm), the chromophore photoconverts predominantly...
  15. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

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    Dec. 23, 2019, 2:33 a.m.
    ...GFP and its homologues have a beta-barrel structure....One of the limitations is the size of GFP: ~240 amino acids or about 28 kDa....Despite these potential issues, GFP has been used successfully in countless fusion proteins....For instance, GFP may occlude catalytic sites or obstruct binding sites or motifs that are necessary...Keep the linker sequence short In the early days of GFP applications, many were concerned with steric...Protein size and shape matters The green fluorescent protein (GFP) from Aequorea victoria and its variants..."A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an...When both orientations are available, a direct comparison of these fusion proteins may reveal which of...The localization of the fusion protein can be verified and it can be compared to what is expected based...
  16. New and Upcoming Viral Vectors - June 2019

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    Jan. 7, 2020, 5:55 p.m.
    ...These samples are available in various serotypes of two popular control AAV encoding GFP.... AAV9, AAVrg pAAV-hSyn-DIO-mCherry 37825 AAV1, 2, 5, 8, 9 (20 µL size serotype testing) pAAV-CAG-GFP...control AAVs Plasmid Serotype Name 105530  AAV1 pAAV.CMV.PI.EGFP.WPRE.bGH 37825  AAV1 pAAV-CAG-GFP...viral vectors New optogenetics inhibitors Plasmid Serotype Name 99039  AAVrg pAAV-CamKII-ArchT-GFP...Controls Plasmid Serotype Name 37825  AAVrg (20 µL size serotype testing) pAAV-CAG-GFP 50465 AAV1...experiments, scientists often have to choose the best serotype for their particular study, which includes comparing...37825  AAV1 pAAV-CAG-GFP 59462  AAV2 pAAV-CAG-tdTomato (codon diversified) 114472  AAV5, AAV8...50465 AAV8, 9 (20 µL size serotype testing) AAV-hSyn-EGFP Biosensor AAV The ability to detect the...(Gq) New chemogenetics AAV Plasmid Serotype Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP...
  17. 22 Hot Plasmid Technologies from 2014

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    Nov. 19, 2019, 3:08 p.m.
    ...Highly bright and extremely dim GFPs The availability of the three dimensional structure of GFP, its...Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown in eyes...on functional complementation between two non-fluorescent split GFP fragments....brighter mutant S65T-GFP, and the subsequent biochemical characterization, has enabled scientists to...mutagenesis in the soil bacterium Pseudomonas putida and demonstrate that they can successfully create GFP...engineer a wide variety of GFPs with diversity in fluorescence brightness, intensity, excitation and...The lab of Dimitri Deheyn recently identified a family of GFP proteins in the cephalochordate Branchiostoma...floridae, named bfloGFPs....The plasmids used for determining the structures are now available from Addgene - bfloGFPa1 and bfloGFPc1...Up to eight entry vectors can be used for each Multisite Gateway compatible destination vector for a...In the first assembly step, compatible Level 0 vectors are directionally assembled into a Level 1 vector...While the DNA-binding specificity is imparted by the RVD at residues 12 and 13 in the TALE repeat, other...
  18. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

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    Oct. 20, 2019, 8:36 p.m.
    ...GFP-tagged Atg8p or LC3: ATG8 is part of a group of genes that affect autophagy (ATG) in yeast....A GFP-tagged version of LC3, the mammalian Atg8p homolog, can also be used to monitor autophagy, but...Atg8p (“p” stands for protein) associates with autophagosomal membranes, so tagging it with GFP allows...two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP...Additional Resources on the Addgene Blog Beware of Fluorescent Protein Oligomerization Seeing Red: Simple GFP...To control for protein degradation due to other pathways, it is standard practice to compare degradation...It’s particularly useful for comparing bulk autophagy vs. mitophagy, the targeted autophagy of mitochondria...autophagy-sensing abilities is that its fluorescence emission spectra changes when it goes from a more neutral pH compartment...
  19. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

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    June 8, 2020, 4:24 p.m.
    ...., bioRxiv 2019. https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity...s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized GFP...type (top panels) and cells with high intracellular Ca2+ (bottom panels) equipped with destabilized GFP...Find the GFP reporter calcineurin activity here Diessl et al., Microbial Cell, 2020. https://dx.doi.org...In comparison to intercalating fluorescent dyes which can cause photo-induced DNA breakage and structural...vector is suitable for flow cytometry and fluorescence microscopy, they also show that this method is compatible...They created plasmids for purification of tTALE-eGFP and tTALE-mCherry....
  20. Inntags: Innovative Protein Epitope Tagging

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    Nov. 22, 2019, 4:32 p.m.
    ...Impressively, IT6-GFP did not reduce yeast cell size or viability compared to untagged GFP overexpression...Using budding yeast as a model system, Georgieva et al. examined the effects of GFP fusions to inntags...Impressively, IT6-GFP did not reduce yeast cell size or viability compared to untagged GFP overexpression...
Showing: 1 - 20 of 152 results