The PACT domain of Drosophila DPLP55 (link) was cloned by PCR from pubiPCTGFP (a gift from J. Raff) using the primer pair 5′-gtacggtaccaacatgattgctctgcagaagaaatg-3′ and 5′-gtacctcgagatgatgccgcgcatgcgctctttttg-3′. The PCR product was subcloned into the pcDNA3 Flag1 EosFP tandem vector that contains the tandem dimer version of the photo-convertible EosFP56 (link) to construct the PACT–d2Eos fusion protein. Using PSI Blast57 (link), the Ensemble Genome Browser and InParanoid, we identified CG5690 as the putative Drosophila ortholog of mouse and human centrobin. We obtained the corresponding full-length cDNA, SD06673, from DGRC (http://dgrc.cgb.indiana.edu), and fused it to EYFP-C1 (Clontec). We subcloned asl47 (link) in the pTagFP635-C vector (mKATE, Evrogen). These three fusion constructs were subcloned into the Drosophila pUBq pWRpUbqu transformation vector, and transgenic lines expressing PACT–d2Eos, mKATE-ASL and YFP–CNB under the control of the poly-Ubiquitin promoter were obtained using standard techniques.
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Amino Acid
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MKate
MKate
MKate is a far-red fluorescent protein derived from the red fluorescent protein eqFP611.
It has an excitation maximum at 588 nm and an emission maximum at 635 nm, making it useful for multicolor imaging and flow cytometry applications.
MKate exhibits high photostability and can be used as a fluorescent tag for proteins, allowing visualization of subcellular structures and dynamic processes in living cells.
Its red-shifted spectrum also enables deeper tissue penetration compared to green fluorescent proteins.
Researchers can leverage the unique properties of MKate to enhance their experinents and advance their scientific investigations.
It has an excitation maximum at 588 nm and an emission maximum at 635 nm, making it useful for multicolor imaging and flow cytometry applications.
MKate exhibits high photostability and can be used as a fluorescent tag for proteins, allowing visualization of subcellular structures and dynamic processes in living cells.
Its red-shifted spectrum also enables deeper tissue penetration compared to green fluorescent proteins.
Researchers can leverage the unique properties of MKate to enhance their experinents and advance their scientific investigations.
Most cited protocols related to «MKate»
Animals, Transgenic
Cloning Vectors
DNA, Complementary
Drosophila
Genome
Homo sapiens
Mice, Laboratory
mKate
Oligonucleotide Primers
Polyubiquitin
Proteins
To generate a rescuing genomic shot transgene with C-terminal YFP tag, we used the PACMAN CH321-44M3 BAC clone (Venken et al., 2009 (link)) covering the entire shot locus. The BAC was modified using the galK positive/counter-selection cassette and recombineering (Warming et al., 2005 (link)). Transgenic flies were created by Genetivision.
The Patronin C-terminal YFP knockin was made by injecting nos>Cas9 embryos (Port et al., 2014 (link)) with a single guide RNA targeting the region of the stop codon in patronin (5′-GGCGCTTGTAATCTAA GCGG-3′, the stop codon is in bold) and a donor plasmid with 4-kb homology arms surrounding the Venus sequence.
pUASP-mKate-ABD was constructed by amplifying Shot ABD and mKate2 with the following primers: 5′-ATGTAGCGGCCGCCCGCGATGCCATTCAGAAGA-3′ and 5′-ATGTATCTAGATCAAATGTACGTGATGAGGGACT-3′; 5′ACGTGGTACCATGGTGAGCGAGCTGATT-3′ and 5′ATGTAGCGGCCGCGGAAGAGGAAGATCTGTGCCCCAGTTTGCT-3′. The amplified fragments were cloned into the pUASP vector (Rørth, 1998 (link)). The mutated Shot ABD was amplified with 5′-GATCAAACTGGACAACATACG-3′ and 5′-CGTATGTTGTCCAGTTTGATC-3′. Shot RE cDNA was obtained from A. Prokop (University of Manchester, UK).
For generation of pUASP-mCherry-Patronin and pUMAT-mCherry-Patronin, patronin RI and mCherry were amplified with 5′-ATGTAGGTACCATGGTGAGCAAGGGCGAGGAGGATAACA-3′ and 5′-GCATTCTAGATTAGATTACAAGCGCCATGTCTTTT-3′ from the pMT-mCherry-Patronin plasmid (Goodwin and Vale, 2010 (link)) (Addgene) and cloned into the pUASP vector (Rørth, 1998 (link)) and the pUMAT vector (Irion et al., 2006 (link)).
For generation of pUMAT-YFP-Patronin, patronin RI and YFP were amplified with 5′-ATGGACGAGCTGTACAAGCACCGGTATACAAGT-3′ and 5′-GCATTCTAGATTAGATTACAAGCGCCATGTCTTTT-3′, and 5′-TAGTAGGTACCCATGAGCAAGGGCGAGG-3′ and 5′-ACTTGTATACCGGTGCTTGTACAGCTCGTCCAT-3′, respectively and cloned into the pUMAT vector (Irion et al., 2006 (link)).
shot2A2 genomic DNA was isolated from homozygous embryos and larvae using the Gentra Puregene Cell Kit (Qiagen), and exonic regions were amplified by PCR and sequenced. Primer sequences are available on request.
The Patronin C-terminal YFP knockin was made by injecting nos>Cas9 embryos (Port et al., 2014 (link)) with a single guide RNA targeting the region of the stop codon in patronin (5′-GGCGCTTGTAATC
pUASP-mKate-ABD was constructed by amplifying Shot ABD and mKate2 with the following primers: 5′-ATGTAGCGGCCGCCCGCGATGCCATTCAGAAGA-3′ and 5′-ATGTATCTAGATCAAATGTACGTGATGAGGGACT-3′; 5′ACGTGGTACCATGGTGAGCGAGCTGATT-3′ and 5′ATGTAGCGGCCGCGGAAGAGGAAGATCTGTGCCCCAGTTTGCT-3′. The amplified fragments were cloned into the pUASP vector (Rørth, 1998 (link)). The mutated Shot ABD was amplified with 5′-GATCAAACTGGACAACATACG-3′ and 5′-CGTATGTTGTCCAGTTTGATC-3′. Shot RE cDNA was obtained from A. Prokop (University of Manchester, UK).
For generation of pUASP-mCherry-Patronin and pUMAT-mCherry-Patronin, patronin RI and mCherry were amplified with 5′-ATGTAGGTACCATGGTGAGCAAGGGCGAGGAGGATAACA-3′ and 5′-GCATTCTAGATTAGATTACAAGCGCCATGTCTTTT-3′ from the pMT-mCherry-Patronin plasmid (Goodwin and Vale, 2010 (link)) (Addgene) and cloned into the pUASP vector (Rørth, 1998 (link)) and the pUMAT vector (Irion et al., 2006 (link)).
For generation of pUMAT-YFP-Patronin, patronin RI and YFP were amplified with 5′-ATGGACGAGCTGTACAAGCACCGGTATACAAGT-3′ and 5′-GCATTCTAGATTAGATTACAAGCGCCATGTCTTTT-3′, and 5′-TAGTAGGTACCCATGAGCAAGGGCGAGG-3′ and 5′-ACTTGTATACCGGTGCTTGTACAGCTCGTCCAT-3′, respectively and cloned into the pUMAT vector (Irion et al., 2006 (link)).
shot2A2 genomic DNA was isolated from homozygous embryos and larvae using the Gentra Puregene Cell Kit (Qiagen), and exonic regions were amplified by PCR and sequenced. Primer sequences are available on request.
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Animals, Transgenic
Arm, Upper
Cells
Cloning Vectors
Codon, Terminator
Diptera
DNA, Complementary
Embryo
Exons
Genome
Homozygote
Larva
mKate
Oligonucleotide Primers
Plasmids
RNA, Single Guide
Tissue Donors
Transgenes
anti-IgG
Antibodies, Anti-Idiotypic
Cells
Chlamydia
Chromosomes
Common Cold
Cytoplasm
Evans Blue
Fluorescein-5-isothiocyanate
Formaldehyde
Glutaral
Goat
HOE 33342
Immunoglobulins
L929 Cells
Lens, Crystalline
Methanol
Microscopy, Confocal
mKate
Proteins
Rabbits
Submersion
Actinin
Adult
Alleles
Amino Acids
Animals, Transgenic
Congenital Heart Defects
Embryo
Genes
Heart
Jumping Genes
mKate
Myocardium
Myocytes, Cardiac
Plasma Membrane
Protein Isoforms
Proteins
red fluorescent protein
Skeletal Muscles
Skeleton
Tissue, Membrane
Transgenes
Zebrafish
All imaging data was quantified in Volocity (PerkinElmer). To find dynamic changes in GFP intensities, cell nuclei were tracked using the LSS2-mKate-PCNA signal and the Shortest Path algorithm. A maximum distance of 9 μm between nuclei in consecutive timeframes was used to eliminate erroneous tracks. For p27Kip1-GFP, CyclinE1-GFP, and CyclinA2-GFP, nuclear levels of protein were quantified since all three proteins had an exclusively nuclear localization during G1 and S. In each cell, the average value of pixel intensities was calculated, and average background fluorescence was subtracted. All fluorescence intensities were normalized to peak levels. Timing of S-phase entry was determined manually from PCNA fluorescence images. Specifically, S-phase entry is defined by a sharp increase in PCNA intensity, often coinciding with the appearance of nucleoli.
For CDK2L-GFP and DHB-Ven quantification, nuclear intensity was measured by taking a region of interest (ROI) inside the nucleus, and cytoplasmic intensity was measured as the ring region around the nucleus. Cdk2 activity was then measured as the ratio between the ring GFP intensity to the nuclear GFP intensity.
Additional experimental procedures are provided inSupplemental Information .
For CDK2L-GFP and DHB-Ven quantification, nuclear intensity was measured by taking a region of interest (ROI) inside the nucleus, and cytoplasmic intensity was measured as the ring region around the nucleus. Cdk2 activity was then measured as the ratio between the ring GFP intensity to the nuclear GFP intensity.
Additional experimental procedures are provided in
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CDK2 protein, human
CDKN1B protein, human
Cell Nucleolus
Cell Nucleus
Cells
Cytoplasm
Fluorescence
mKate
Nuclear Protein
Proliferating Cell Nuclear Antigen
Proteins
Most recents protocols related to «MKate»
PCR fragments from pBG42 (34) and pBG42 mKate were designed with overhangs homologous to mini-ctx2 (35) using PCR. These fragments contain aacC1 (gentamycin resistance) and msfGFP or mKate. Plasmid preparation of mini-ctx2 was digested using SacI (Thermofisher) and purified. RecET recombineering was used to construct mini-ctx2 aacC1 msfGFP and mini-ctx2 aacC1 mKate. These plasmids integrate into the CTX attachment site in the P. aeruginosa genome and thus mediate genomic tagging of P. aeruginosa with aacC1 and a green or red fluorescent marker. The plasmids were introduced into P. aeruginosa by conjugation according to Hoang et al (35) .
The pUASTattB-dAtad3R472C-V5 construct was generated by performing site-directed mutagenesis PCR using pUASTattB-dAtad3a WT-V521 (link) as a template and the following primers: (R472C)F: 5′-tttgattatgccatcaacgatTGC ctggatgaaatggtggagttc-3′, (R472C) R: 5′-gaactccaccatttcatccagGCA atcgttgatggcataatcaaa-3′. For the construction of pUASTattB-mKate-ThetaTox-D4, we amplified mKate-ThetaTox-D4 coding sequence from pAAV-GFAP-mKate-FL-ThetaTox, using the following primers: mKate-D4_F: 5′-attttgAGATCTcaaa ATG GAGCTGATTAAGGAGAACATG-3′; and mKate-D4_R: 5′-aactaaGCGGCCGC TTAGTTGTAGGTGATGCTGCT-3′.
The amplified PCR product was cloned into the BglII and NotI sites of pUASTattB vector.29 (link) The DNA clones were amplified and purified by the PureLink® HiPure Plasmid Midiprep Kits (Invitrogen). The sequences of mid-prep DNA clones were verified by Sanger sequencing and injected into the following embryos: pUASTattB-mKate-ThetaTox-D4 (y1 w1118; PBac{y+-attP-3B}VK00033); and pUASTattB-dAtad3R472C-V5 (y1 w1118; PBac{y+-attP-3B}VK00037).30 (link) Transgenic flies were selected with a W+ marker and balanced.
The insc-Gal4 driver (w*; P{GawB}inscMz1407; # 8751) and UAS-GFP-LAMP (on the second chromosome; 42714) were obtained from the Bloomington Drosophila Stock Center at Indiana University (BDSC). dAtad3 RNAi line (v22445) was obtained from VDRC stock centre. To generate flies carrying both insc-Gal4 and UAS-GFP-LAMP, we performed recombination of these two genetic components. The flies carrying insc-Gal4, UAS-GFP-LAMP were verified by genomic PCR using the following primers. pUAST-F: 5′-AGTGCAAGTTAAAGTGAATC-3′ EGFP-R: 5′-CGCCTTCTTGACGAGTTCTTC-3′.
To determine the effects of dAtad3R472C expression on the levels of cholesterol-containing membranes and lysosomes in the Drosophila neuroblasts, we crossed the w*; insc-Gal4, UAS-GFP-Lamp flies with the flies carrying y, w; UAS-dAtad3R472C-V5; UAS-mKate-ThetaTox-D4. For control, we used files carrying UAS-empty31 (link) and UAS-mKate-ThetaTox-D4.
All flies were maintained at room temperature (21°C). All crosses were maintained at 25°C. One litre of the standard diet comprised 45.45 g cornmeal, 9.1 g soy flour, 15.4 g yeast, 100 ml syrup, 6.8 g agar, 4.27 ml propionic acid; while the modified diets were as follows: modified diet (MD)—22.72 g cornmeal, 4.55 g soy flour, 7.7 g yeast, 135 ml syrup, 6.8 g agar, 4.27 ml propionic acid; MD2—11.36 g cornmeal, 2.27 g soy flour, 3.85 g yeast, 152.5 ml syrup, 6.8 g agar, 4.27 ml propionic acid, with or without 0.1 or 1 g/l cholesterol (0433 VWR).
The amplified PCR product was cloned into the BglII and NotI sites of pUASTattB vector.29 (link) The DNA clones were amplified and purified by the PureLink® HiPure Plasmid Midiprep Kits (Invitrogen). The sequences of mid-prep DNA clones were verified by Sanger sequencing and injected into the following embryos: pUASTattB-mKate-ThetaTox-D4 (y1 w1118; PBac{y+-attP-3B}VK00033); and pUASTattB-dAtad3R472C-V5 (y1 w1118; PBac{y+-attP-3B}VK00037).30 (link) Transgenic flies were selected with a W+ marker and balanced.
The insc-Gal4 driver (w*; P{GawB}inscMz1407; # 8751) and UAS-GFP-LAMP (on the second chromosome; 42714) were obtained from the Bloomington Drosophila Stock Center at Indiana University (BDSC). dAtad3 RNAi line (v22445) was obtained from VDRC stock centre. To generate flies carrying both insc-Gal4 and UAS-GFP-LAMP, we performed recombination of these two genetic components. The flies carrying insc-Gal4, UAS-GFP-LAMP were verified by genomic PCR using the following primers. pUAST-F: 5′-AGTGCAAGTTAAAGTGAATC-3′ EGFP-R: 5′-CGCCTTCTTGACGAGTTCTTC-3′.
To determine the effects of dAtad3R472C expression on the levels of cholesterol-containing membranes and lysosomes in the Drosophila neuroblasts, we crossed the w*; insc-Gal4, UAS-GFP-Lamp flies with the flies carrying y, w; UAS-dAtad3R472C-V5; UAS-mKate-ThetaTox-D4. For control, we used files carrying UAS-empty31 (link) and UAS-mKate-ThetaTox-D4.
All flies were maintained at room temperature (21°C). All crosses were maintained at 25°C. One litre of the standard diet comprised 45.45 g cornmeal, 9.1 g soy flour, 15.4 g yeast, 100 ml syrup, 6.8 g agar, 4.27 ml propionic acid; while the modified diets were as follows: modified diet (MD)—22.72 g cornmeal, 4.55 g soy flour, 7.7 g yeast, 135 ml syrup, 6.8 g agar, 4.27 ml propionic acid; MD2—11.36 g cornmeal, 2.27 g soy flour, 3.85 g yeast, 152.5 ml syrup, 6.8 g agar, 4.27 ml propionic acid, with or without 0.1 or 1 g/l cholesterol (0433 VWR).
Strains with the promoter from the RhlR-regulated gene rhlI fused to green fluorescent protein and the lacZ gene were grown in monoculture or in co-culture with a “wild-type” strain constitutively expressing the mKate2 fluorophore under a synthetic tac promoter, referred to as the PA14 att:Ptac-mKate strain. Overnight LB cultures of the PA14 att:Ptac-mKate strain and the designated lasR mutants with the lacZ–GFP fusion integrated at the Tn7 att site were adjusted to an OD600 of 1. For monocultures, a 250-µL aliquot was added to 5 mL fresh LB. For co-cultures, 200 µL of the PA14 att:Ptac-mKate and 50 µL of the designated lasR mutant were added to 5 mL of fresh LB. After 6 h of incubation on a roller drum at 37°C, a 500-µL aliquot of the subculture was pelleted for 5 min at 13,000 RPM, resuspended in 500 µL of PBS + 0.01% Triton X-100, diluted 100-fold in PBS + 0.01% Triton X-100, and diluted again 1:1 in PBS without detergent. The diluted cells were placed on ice until processed by flow cytometry. The data were collected by Beckman Coulter Cytoflex S and analyzed with FlowJo version 10.8.1. In short, single-cell gating was done using FSC versus SSC, and cells without mKate expression were gated using the ECD channel. GFP expression was quantified by fluorescein isothiocyanate (FITC)median fluorescence intensity of the single-cell, mKate-negative populations.
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On day -1, 1250 266-6-mKate cells were seeded in 96F plate in DMEM media supplemented with 10% FCS, 100 U/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco) (Supplementary Figure 5G). On day 0, Treg cells (Live CD45 + CD3 + CD8 - CD4 + TCRβ + Foxp3 YFP+ ) and Tconv cells (Live CD45 + CD3 + CD8 -CD4 + TCRβ + Foxp3 YFP-) were isolated by flow cytometry from single cell suspensions prepared from pancreata of IL-33treated Foxp3 YFP mice using a FACS Aria instrument. 3000 purified T cells in RPMI supplemented with 2.5%FCS were added to pre-washed 266-6 mKate cells, along with rmIL-33 (20 ng/ml, Biolegend), rhIL-2 (50 ng/ml, Peprotech), and Dynabeads Mouse T-Activator CD3/CD28 (2 beads:1 cell ratio, Themo Scientific), followed by incubation in the Incucyte live imaging system for measurement of 266.6-mKate proliferation.
Homologues of the BoYgl-2 protein were obtained from the National Center for Biotechnology Information (NCBI) database by a BLASTP search. Phylogenetic analysis was performed as described previously [32 (link)].
The full-length CDS of BoYgl-2 without the stop codon was cloned into the green fluorescent protein (GFP) expression vector pBWA(V)HS-35S-GFP. Far red fluorescent protein (mKate) expression vector was used as the nuclear marker. The fusion construct and the mKate construct were transformed into Agrobacterium GV3101 and then injected into tobacco leaves as described previously [5 (link)]. GFP fluorescence signals were detected under a confocal laser-scanning microscope (Carl Zeiss, Germany).
The full-length CDS of BoYgl-2 without the stop codon was cloned into the green fluorescent protein (GFP) expression vector pBWA(V)HS-35S-GFP. Far red fluorescent protein (mKate) expression vector was used as the nuclear marker. The fusion construct and the mKate construct were transformed into Agrobacterium GV3101 and then injected into tobacco leaves as described previously [5 (link)]. GFP fluorescence signals were detected under a confocal laser-scanning microscope (Carl Zeiss, Germany).
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More about "MKate"
MKate is a far-red fluorescent protein that has become a valuable tool in scientific research, particularly in multicolor imaging and flow cytometry applications.
Derived from the red fluorescent protein eqFP611, MKate exhibits an excitation maximum at 588 nm and an emission maximum at 635 nm, making it well-suited for deep tissue penetration and visualization of subcellular structures and dynamic processes in living cells.
One of the key advantages of MKate is its high photostability, which allows researchers to capture high-quality images and videos of their experiments over extended periods.
This property, combined with its red-shifted spectrum, makes MKate an attractive alternative to traditional green fluorescent proteins (GFPs) for a variety of applications.
For example, MKate can be used as a fluorescent tag for proteins, enabling scientists to track the localization and movement of specific cellular components using techniques like confocal microscopy (LSM 710) and flow cytometry (Cytoflex S).
This can be particularly useful for investigating complex biological processes, such as signaling pathways, organelle dynamics, and cell-cell interactions.
Beyond its applications in imaging and flow cytometry, MKate's unique properties can also be leveraged in other experimental setups.
Researchers may use MKate-tagged proteins in combination with other fluorescent probes, such as EMCCD cameras and SpectraMax Paradigm or SpectraMax M2e plate readers, to perform multicolor analyses and gain a more comprehensive understanding of their systems.
Additionally, MKate can be expressed in a variety of cell types, including mammalian cells, yeast, and bacteria, making it a versatile tool for a wide range of experiments.
Researchers may use transfection reagents like Lipofectamine 3000 or antibiotics like Penicillin/Streptomycin to introduce MKate-encoding plasmids into their cell lines and study the resulting effects.
Ultimately, the versatility and performance of MKate have made it an invaluable resource for scientists across various fields, from cell biology and neuroscience to immunology and synthetic biology.
By leveraging the unique properties of this far-red fluorescent protein, researchers can enhance their experiments, uncover new insights, and advance their scientific investigations.
Derived from the red fluorescent protein eqFP611, MKate exhibits an excitation maximum at 588 nm and an emission maximum at 635 nm, making it well-suited for deep tissue penetration and visualization of subcellular structures and dynamic processes in living cells.
One of the key advantages of MKate is its high photostability, which allows researchers to capture high-quality images and videos of their experiments over extended periods.
This property, combined with its red-shifted spectrum, makes MKate an attractive alternative to traditional green fluorescent proteins (GFPs) for a variety of applications.
For example, MKate can be used as a fluorescent tag for proteins, enabling scientists to track the localization and movement of specific cellular components using techniques like confocal microscopy (LSM 710) and flow cytometry (Cytoflex S).
This can be particularly useful for investigating complex biological processes, such as signaling pathways, organelle dynamics, and cell-cell interactions.
Beyond its applications in imaging and flow cytometry, MKate's unique properties can also be leveraged in other experimental setups.
Researchers may use MKate-tagged proteins in combination with other fluorescent probes, such as EMCCD cameras and SpectraMax Paradigm or SpectraMax M2e plate readers, to perform multicolor analyses and gain a more comprehensive understanding of their systems.
Additionally, MKate can be expressed in a variety of cell types, including mammalian cells, yeast, and bacteria, making it a versatile tool for a wide range of experiments.
Researchers may use transfection reagents like Lipofectamine 3000 or antibiotics like Penicillin/Streptomycin to introduce MKate-encoding plasmids into their cell lines and study the resulting effects.
Ultimately, the versatility and performance of MKate have made it an invaluable resource for scientists across various fields, from cell biology and neuroscience to immunology and synthetic biology.
By leveraging the unique properties of this far-red fluorescent protein, researchers can enhance their experiments, uncover new insights, and advance their scientific investigations.