biotinylated dna fragments  (Thermo Fisher)


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    Name:
    Dynabeads MyOne Streptavidin C1
    Description:
    Uniform and superparamagnetic Dynabeads 1 µm in diameter with a monolayer of recombinant streptavidin covalently coupled to the surface The hydrophilic and negatively charged beads allow for efficient isolation and downstream handling of your biotinylated nucleic acids antibodies or other biotinylated ligand or target molecules The streptavidin monolayer ensures negligible leakage and the lack of excess adsorbed streptavidin ensures batch consistency and reproducibility of your results The 1 µm MyOne beads are widely used as a solid phase in automated protocols where high throughput is crucial The liquid phase behavior in combination with the superparamagnetic properties of the beads provide rapid reaction kinetics both in the coating process separation and during washing of the analyte The beads also feature a large surface area high capacity efficient magnetic pull and a slow sedimentation rate during incubation The product holds reputable Dynal high standards with respect to reproducibility and automation ability and drives reliability for your assays Benefits and Features • Direct and fast capture of any biotinylated ligand or target• Flexible solid phase protocols with superior liquid phase reaction kinetics• Small bead size high capacity and improved reaction kinetics compared to the M 280⁄M 270 beads• Low sedimention rate yet a high iron content ensuring rapid magnetic separation• Easy and reproducible handling in manufacturing• Very low aggregation• Fast and efficient washing procedures• Low non specific binding of small and negatively charged proteins• Very low non specific binding of nucleotides and nucleic acids• Well suited for nucleic acid applications with extreme demands• Reproducible behavior in automation without mixing requirements• High batch to batch reproducibility securing consistent results in your application• Production follows a validated process in compliance with cGMP for medical deviceApplications Efficient capture of biotinylated molecules For direct⁄indirect isolation and downstream handling of nucleic acids proteins⁄peptides and other target molecules Ideal for sequence specific DNA⁄RNA capture in nucleic acid based diagnostics specifically with samples with a high chaotropic salt concentration immunoassays involving small biotinylated antigens and applications that are not compatible with BSA Well suited for immunodiagnostics with hydrophobic targets Binding capacity The size of the molecule and the biotinylation procedure will affect the binding capacity The capacity also depends on steric availability and charge interaction between bead and molecule and between molecules There are two or three biotin binding sites available for each streptavidin molecule on the surface of the bead after immobilization One mg of Dynabeads M 280 Streptavidin typically binds 950 pmoles free biotin approx 200 pmol biotinylated peptides up to10 µg biotinylated antibody approx 10 µg ds DNA or 200 pmol ss Oligonucleotides Additional Info This specific product format is for large volume customers available on an OEM basis The product is also available in smaller volumes for end users Cat no 650 01 650 02 and 650 03
    Catalog Number:
    35002D
    Price:
    None
    Category:
    Beads Microspheres
    Applications:
    Bead-Based IVD Assay Development|Bead-Based Immunoassay IVD|Bead-Based Nucleic Acid IVD|Clinical|DNA & RNA Purification & Analysis|DNA Extraction|Diagnostic Development|Molecular Diagnostic Test Development|Sequence-Specific DNA or RNA Purification
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    Structured Review

    Thermo Fisher biotinylated dna fragments
    RPA binding to ICL <t>DNA</t> is dependent on FAAP24 (A) Migration of ICL DNA on a denaturing gel. (B) Immunoblot showing that FAAP24 wild-type (WT) but not the C-terminal truncated FAAP24 mutant (N150) binds to ICL DNA. <t>Biotinylated</t> control DNA or ICL-DNA (100 ng) were attached to streptavidin-coated beads and incubated with purified His-tagged WT FAAP24 or N150 FAAP24. (C) Immunoblot showing that RPA loading to ICL DNA was decreased in FAAP24-depleted nuclear extracts. Nuclear extracts (100 μg) derived from HeLa scramble or FAAP24 shRNA cells were incubated with biotinylated control DNA or ICL-DNA (100 ng) respectively. The DNA-bound FAAP24, RPA2 and KU70 were detected. (D) Proposed working model for the role of FANCM/FAAP24 in the ICL-induced checkpoint response.
    Uniform and superparamagnetic Dynabeads 1 µm in diameter with a monolayer of recombinant streptavidin covalently coupled to the surface The hydrophilic and negatively charged beads allow for efficient isolation and downstream handling of your biotinylated nucleic acids antibodies or other biotinylated ligand or target molecules The streptavidin monolayer ensures negligible leakage and the lack of excess adsorbed streptavidin ensures batch consistency and reproducibility of your results The 1 µm MyOne beads are widely used as a solid phase in automated protocols where high throughput is crucial The liquid phase behavior in combination with the superparamagnetic properties of the beads provide rapid reaction kinetics both in the coating process separation and during washing of the analyte The beads also feature a large surface area high capacity efficient magnetic pull and a slow sedimentation rate during incubation The product holds reputable Dynal high standards with respect to reproducibility and automation ability and drives reliability for your assays Benefits and Features • Direct and fast capture of any biotinylated ligand or target• Flexible solid phase protocols with superior liquid phase reaction kinetics• Small bead size high capacity and improved reaction kinetics compared to the M 280⁄M 270 beads• Low sedimention rate yet a high iron content ensuring rapid magnetic separation• Easy and reproducible handling in manufacturing• Very low aggregation• Fast and efficient washing procedures• Low non specific binding of small and negatively charged proteins• Very low non specific binding of nucleotides and nucleic acids• Well suited for nucleic acid applications with extreme demands• Reproducible behavior in automation without mixing requirements• High batch to batch reproducibility securing consistent results in your application• Production follows a validated process in compliance with cGMP for medical deviceApplications Efficient capture of biotinylated molecules For direct⁄indirect isolation and downstream handling of nucleic acids proteins⁄peptides and other target molecules Ideal for sequence specific DNA⁄RNA capture in nucleic acid based diagnostics specifically with samples with a high chaotropic salt concentration immunoassays involving small biotinylated antigens and applications that are not compatible with BSA Well suited for immunodiagnostics with hydrophobic targets Binding capacity The size of the molecule and the biotinylation procedure will affect the binding capacity The capacity also depends on steric availability and charge interaction between bead and molecule and between molecules There are two or three biotin binding sites available for each streptavidin molecule on the surface of the bead after immobilization One mg of Dynabeads M 280 Streptavidin typically binds 950 pmoles free biotin approx 200 pmol biotinylated peptides up to10 µg biotinylated antibody approx 10 µg ds DNA or 200 pmol ss Oligonucleotides Additional Info This specific product format is for large volume customers available on an OEM basis The product is also available in smaller volumes for end users Cat no 650 01 650 02 and 650 03
    https://www.bioz.com/result/biotinylated dna fragments/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated dna fragments - by Bioz Stars, 2021-04
    86/100 stars

    Images

    1) Product Images from "The FANCM/FAAP24 Complex is Required for the DNA Inter-strand Crosslink-Induced Checkpoint Response"

    Article Title: The FANCM/FAAP24 Complex is Required for the DNA Inter-strand Crosslink-Induced Checkpoint Response

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2010.07.005

    RPA binding to ICL DNA is dependent on FAAP24 (A) Migration of ICL DNA on a denaturing gel. (B) Immunoblot showing that FAAP24 wild-type (WT) but not the C-terminal truncated FAAP24 mutant (N150) binds to ICL DNA. Biotinylated control DNA or ICL-DNA (100 ng) were attached to streptavidin-coated beads and incubated with purified His-tagged WT FAAP24 or N150 FAAP24. (C) Immunoblot showing that RPA loading to ICL DNA was decreased in FAAP24-depleted nuclear extracts. Nuclear extracts (100 μg) derived from HeLa scramble or FAAP24 shRNA cells were incubated with biotinylated control DNA or ICL-DNA (100 ng) respectively. The DNA-bound FAAP24, RPA2 and KU70 were detected. (D) Proposed working model for the role of FANCM/FAAP24 in the ICL-induced checkpoint response.
    Figure Legend Snippet: RPA binding to ICL DNA is dependent on FAAP24 (A) Migration of ICL DNA on a denaturing gel. (B) Immunoblot showing that FAAP24 wild-type (WT) but not the C-terminal truncated FAAP24 mutant (N150) binds to ICL DNA. Biotinylated control DNA or ICL-DNA (100 ng) were attached to streptavidin-coated beads and incubated with purified His-tagged WT FAAP24 or N150 FAAP24. (C) Immunoblot showing that RPA loading to ICL DNA was decreased in FAAP24-depleted nuclear extracts. Nuclear extracts (100 μg) derived from HeLa scramble or FAAP24 shRNA cells were incubated with biotinylated control DNA or ICL-DNA (100 ng) respectively. The DNA-bound FAAP24, RPA2 and KU70 were detected. (D) Proposed working model for the role of FANCM/FAAP24 in the ICL-induced checkpoint response.

    Techniques Used: Recombinase Polymerase Amplification, Binding Assay, Migration, Mutagenesis, Incubation, Purification, Derivative Assay, shRNA

    2) Product Images from "Mobile element scanning (ME-Scan) identifies thousands of novel Alu insertions in diverse human populations"

    Article Title: Mobile element scanning (ME-Scan) identifies thousands of novel Alu insertions in diverse human populations

    Journal: Genome Research

    doi: 10.1101/gr.148973.112

    ME-Scan library preparation ( A–E ) and sequencing ( F–H ). ( A ) Genomic DNA (green) containing an Alu Yb8/9 (blue) in the forward orientation. The 5′ GC-rich region, the 3′ poly-A tail, and the Alu Yb8/9-specific primer site ( Alu BP2) are shown as darker segments. The target site duplications (TSD) are indicated by boxed black arrows. ( B ) DNA samples are fragmented, end-repaired, and an overhanging 3′ A is added. ( C ) Partially double-stranded oligonucleotide adapters (orange) with 9-bp indexes (darker segment) are ligated onto fragment ends. Indexed samples are then pooled. ( D ) Alu Yb8/9 element junctions are targeted by PCR using a biotinylated Alu Yb8/9-specific primer ( Alu BP2) and adapter primer PEP2. Biotinylated DNA molecules are then purified using streptavidin-coated paramagnetic beads. ( E ) Reamplification of the library with primers PEP1 and PEP2 (orange arrows). ( F–H ) The pooled junction library is then sequenced with a three-read design. Primers are shown as arrows, sequencing reads as dashed lines. The first sequencing read (50 nt) extends from Alu SPv2 ( F ) or Alu SPv3 ( G ). To skip the 5′ end of the Alu insertions, which would be identical over the entire flow cell and therefore difficult for Illumina's software to process, the first 30 cycles of nucleotide synthesis are carried out without collecting data (represented by a dashed gray ‘hop’). The second read proceeds for 57 bp from Alu SPIn1. The third read is generated using Illumina's standard second-end read primer and consists of a 9-bp index, a ‘T,’ and 36 bp of genomic sequence from a fragmentation site 50–300 bp upstream of the Alu insertion. Read sets are generated from multiple different fragments representing each Alu Yb8/9 insertion in the library ( H ). Each insertion is uniquely identified by its “ Alu Junction Position” (dashed line and large arrow in F and G ).
    Figure Legend Snippet: ME-Scan library preparation ( A–E ) and sequencing ( F–H ). ( A ) Genomic DNA (green) containing an Alu Yb8/9 (blue) in the forward orientation. The 5′ GC-rich region, the 3′ poly-A tail, and the Alu Yb8/9-specific primer site ( Alu BP2) are shown as darker segments. The target site duplications (TSD) are indicated by boxed black arrows. ( B ) DNA samples are fragmented, end-repaired, and an overhanging 3′ A is added. ( C ) Partially double-stranded oligonucleotide adapters (orange) with 9-bp indexes (darker segment) are ligated onto fragment ends. Indexed samples are then pooled. ( D ) Alu Yb8/9 element junctions are targeted by PCR using a biotinylated Alu Yb8/9-specific primer ( Alu BP2) and adapter primer PEP2. Biotinylated DNA molecules are then purified using streptavidin-coated paramagnetic beads. ( E ) Reamplification of the library with primers PEP1 and PEP2 (orange arrows). ( F–H ) The pooled junction library is then sequenced with a three-read design. Primers are shown as arrows, sequencing reads as dashed lines. The first sequencing read (50 nt) extends from Alu SPv2 ( F ) or Alu SPv3 ( G ). To skip the 5′ end of the Alu insertions, which would be identical over the entire flow cell and therefore difficult for Illumina's software to process, the first 30 cycles of nucleotide synthesis are carried out without collecting data (represented by a dashed gray ‘hop’). The second read proceeds for 57 bp from Alu SPIn1. The third read is generated using Illumina's standard second-end read primer and consists of a 9-bp index, a ‘T,’ and 36 bp of genomic sequence from a fragmentation site 50–300 bp upstream of the Alu insertion. Read sets are generated from multiple different fragments representing each Alu Yb8/9 insertion in the library ( H ). Each insertion is uniquely identified by its “ Alu Junction Position” (dashed line and large arrow in F and G ).

    Techniques Used: Sequencing, Polymerase Chain Reaction, Purification, Flow Cytometry, Software, Generated

    3) Product Images from "Mechanisms of precise genome editing using oligonucleotide donors"

    Article Title: Mechanisms of precise genome editing using oligonucleotide donors

    Journal: Genome Research

    doi: 10.1101/gr.214775.116

    The biotin pull-down assay. ( A , B ) Schematic illustration of the biotin pull-down assay via the SDSA ( A ) and ssDI ( B ) pathways. In the case of ssDI ( B ), the biotinylated ODN is predicted to be incorporated into the target genomic locus, whereas in SDSA ( A ), it should not. The XhoI- and XbaI-digested genomic fragments covalently linked to biotin can be enriched using streptavidin beads under denaturing conditions. The primers BFP_QF and BFP_QR can specifically amplify these genomic fragments with ODN incorporation but not free ODN donors. Biotin, yellow circles; streptavidin, orange ovals; genomic DNA, black lines; ODN sequence, solid red lines; DNA synthesis, dashed red lines; chromophore sequence, TY and SH; genomic lesions, hatched orange lines; homology regions, dashed silver crosses; restriction sites, XhoI and XbaI; PCR primers, horizontal arrows. ( C ), and detected on an agarose gel.
    Figure Legend Snippet: The biotin pull-down assay. ( A , B ) Schematic illustration of the biotin pull-down assay via the SDSA ( A ) and ssDI ( B ) pathways. In the case of ssDI ( B ), the biotinylated ODN is predicted to be incorporated into the target genomic locus, whereas in SDSA ( A ), it should not. The XhoI- and XbaI-digested genomic fragments covalently linked to biotin can be enriched using streptavidin beads under denaturing conditions. The primers BFP_QF and BFP_QR can specifically amplify these genomic fragments with ODN incorporation but not free ODN donors. Biotin, yellow circles; streptavidin, orange ovals; genomic DNA, black lines; ODN sequence, solid red lines; DNA synthesis, dashed red lines; chromophore sequence, TY and SH; genomic lesions, hatched orange lines; homology regions, dashed silver crosses; restriction sites, XhoI and XbaI; PCR primers, horizontal arrows. ( C ), and detected on an agarose gel.

    Techniques Used: Pull Down Assay, Sequencing, DNA Synthesis, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    4) Product Images from "CcpA and LacD.1 Affect Temporal Regulation of Streptococcus pyogenes Virulence Genes ▿ Virulence Genes ▿ †"

    Article Title: CcpA and LacD.1 Affect Temporal Regulation of Streptococcus pyogenes Virulence Genes ▿ Virulence Genes ▿ †

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00746-09

    CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for biotinylated DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of
    Figure Legend Snippet: CcpA binds to a conserved CRE site in the lctO promoter. (A) Diagram of the lctO locus and DNA probes generated by PCR for biotinylated DNA probe precipitations. Positions of DNA segments are shown as relative to the predicted start of translation of

    Techniques Used: Generated, Polymerase Chain Reaction

    5) Product Images from "Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake"

    Article Title: Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake

    Journal: mBio

    doi: 10.1128/mBio.02141-14

    NtrX regulates putA and glnA expression. (A) EMSA for rNtrX binding to the promoter regions of putA (left) and glnA (right). The length (bp) of the probe is shown above each panel. For each panel, biotinylated DNA probe (2 nM) was incubated alone (P), with rNtrX (10 nM, lane 1), or with rNtrX in the presence of 50-fold excess of the corresponding unlabeled DNA competitor (lane 2). Shifted bands are indicated by arrowheads. (B) EMSA for dose-dependent binding of rNtrX and rNtrX-P to the putA promoter. Biotinylated DNA probe (2 nM) was incubated alone (P) or with rNtrX or rNtrX-P at different concentrations (lanes 1 to 3: 2, 4, and 8 nM, respectively). Shifted bands are indicated by an arrowhead. Black triangles show proportions of protein amounts. The numbers below the panel indicate the percentage of bound probe with respect to the total of each lane. (C) EMSA for dose-dependent binding of rNtrX and rNtrX-P to the glnA promoter. Biotinylated DNA probe (2 nM) was incubated alone (P) or with rNtrX at different concentrations (lanes 1 to 3: 6, 20, and 60 nM, respectively) or with rNtrX-P at different concentrations (lanes 1 to 3: 1.5, 5, and 15 nM, respectively). Shifted bands are indicated by an arrowhead. Black triangles show proportions of protein amounts. The numbers below the panel indicate the percentage of bound probe with respect to the total of each lane. (D) NtrX transactivates glnA and putA promoter- lacZ fusions in E. coli . E. coli strains containing pET-33b(+) encoding rNtrX and rNtrYHKD were transformed with the glnA -, putA -, or ompA-lacZ fusions containing the promoter regions of glnA (243 bp), putA (163 bp), or ompA (368 bp), respectively. After induction of rNtrX or rNtrYHKD with IPTG, β-galactosidase activity was measured. Top, β-galactosidase activity (Miller units). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *, significantly different ( P
    Figure Legend Snippet: NtrX regulates putA and glnA expression. (A) EMSA for rNtrX binding to the promoter regions of putA (left) and glnA (right). The length (bp) of the probe is shown above each panel. For each panel, biotinylated DNA probe (2 nM) was incubated alone (P), with rNtrX (10 nM, lane 1), or with rNtrX in the presence of 50-fold excess of the corresponding unlabeled DNA competitor (lane 2). Shifted bands are indicated by arrowheads. (B) EMSA for dose-dependent binding of rNtrX and rNtrX-P to the putA promoter. Biotinylated DNA probe (2 nM) was incubated alone (P) or with rNtrX or rNtrX-P at different concentrations (lanes 1 to 3: 2, 4, and 8 nM, respectively). Shifted bands are indicated by an arrowhead. Black triangles show proportions of protein amounts. The numbers below the panel indicate the percentage of bound probe with respect to the total of each lane. (C) EMSA for dose-dependent binding of rNtrX and rNtrX-P to the glnA promoter. Biotinylated DNA probe (2 nM) was incubated alone (P) or with rNtrX at different concentrations (lanes 1 to 3: 6, 20, and 60 nM, respectively) or with rNtrX-P at different concentrations (lanes 1 to 3: 1.5, 5, and 15 nM, respectively). Shifted bands are indicated by an arrowhead. Black triangles show proportions of protein amounts. The numbers below the panel indicate the percentage of bound probe with respect to the total of each lane. (D) NtrX transactivates glnA and putA promoter- lacZ fusions in E. coli . E. coli strains containing pET-33b(+) encoding rNtrX and rNtrYHKD were transformed with the glnA -, putA -, or ompA-lacZ fusions containing the promoter regions of glnA (243 bp), putA (163 bp), or ompA (368 bp), respectively. After induction of rNtrX or rNtrYHKD with IPTG, β-galactosidase activity was measured. Top, β-galactosidase activity (Miller units). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *, significantly different ( P

    Techniques Used: Expressing, Binding Assay, Incubation, Positron Emission Tomography, Transformation Assay, Activity Assay

    6) Product Images from "GATA binding protein 3 is correlated with leptin regulation of PPARγ1 in hepatic stellate cells"

    Article Title: GATA binding protein 3 is correlated with leptin regulation of PPARγ1 in hepatic stellate cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13002

    GATA 3 binds to GATA 2‐binding site on PPAR γ1 promoter and interacts with GATA 2. ( A ) EMSA of GATA 3 binding to GATA 2‐binding site on PPAR γ1 promoter ( n = 3). After HSC s were stimulated with 100 ng/ml of leptin for 24 hrs, nuclear extracts ( NE ) were prepared and 5 μg of nuclear proteins were incubated with biotinylated DNA fragment (containing the GATA ‐2‐binding site around −2323). For competition assay, 5 μg of nuclear proteins were preincubated with 100‐fold molar excess of the unlabelled probe before addition of labelled probe. For supershift assay, 5 μg of nuclear proteins were preincubated with 1 μg of anti‐ GATA 3 antibody, or 1 μg of anti‐ GATA 2 antibody, or 1 μg of normal IgG, or 0.5 μg of anti‐ GATA 3 antibody plus 0.5 μg of anti‐ GATA 2 antibody before addition of the labelled probe. A representative EMSA was shown. ( B ) Ch IP analysis of GATA 3 binding to the site (around −2323) of PPAR γ1 promoter ( n = 3). HSC s were stimulated with leptin or the vehicle (−) for 24 hrs and Ch IP analysis was performed by using anti‐ GATA 3 antibody or normal IgG as described in materials and methods. The purified DNA from immunoprecipitation and from the input samples were used to amplify a fragment (132 bp) between −2362 and −2230 (containing the GATA ‐2‐binding site) by PCR and the PCR products were examined by 2% agarose gel electrophoresis. ( C ) Immunoprecipitation assay of the interaction between GATA 3 and GATA 2 ( n = 3). After pF lag‐ GATA 3 (encoding Flag‐ GATA 3 protein) were cotransfected 293T cells with pc DNAGATA 2 (encoding mouse GATA 2 protein) and incubated for 24 hrs, 293T cells were lysed by RIPA buffer and the some of the cell lysate was used as input sample and the others were used for immunoprecipitation by using the anti‐Flag antibody or the normal IgG. Precipitated immune complexes were detected by western blot analysis by using antibody against GATA 2 or GATA 3. A representative result was shown. ( D ) Luciferase assay ( n = 3). The first group of HSC s were cotransfected with 0.8 μg of pPPAR γ1( GATA mut)Luc or 0.8 μg of pPPAR γ1(−2333)Luc plus 0.8 μg of pc DNAGATA 3 or 0.8 μg of the empty vector and then incubated for 24 hrs. The second group of HSC s were cotransfected with 0.8 μg of p3× GATAL uc or pGL 3‐promoter vector (control) plus 0.8 μg of pc DNAGATA 3 or empty vector and then incubated for 24 hrs. Luciferase assay was performed. * P
    Figure Legend Snippet: GATA 3 binds to GATA 2‐binding site on PPAR γ1 promoter and interacts with GATA 2. ( A ) EMSA of GATA 3 binding to GATA 2‐binding site on PPAR γ1 promoter ( n = 3). After HSC s were stimulated with 100 ng/ml of leptin for 24 hrs, nuclear extracts ( NE ) were prepared and 5 μg of nuclear proteins were incubated with biotinylated DNA fragment (containing the GATA ‐2‐binding site around −2323). For competition assay, 5 μg of nuclear proteins were preincubated with 100‐fold molar excess of the unlabelled probe before addition of labelled probe. For supershift assay, 5 μg of nuclear proteins were preincubated with 1 μg of anti‐ GATA 3 antibody, or 1 μg of anti‐ GATA 2 antibody, or 1 μg of normal IgG, or 0.5 μg of anti‐ GATA 3 antibody plus 0.5 μg of anti‐ GATA 2 antibody before addition of the labelled probe. A representative EMSA was shown. ( B ) Ch IP analysis of GATA 3 binding to the site (around −2323) of PPAR γ1 promoter ( n = 3). HSC s were stimulated with leptin or the vehicle (−) for 24 hrs and Ch IP analysis was performed by using anti‐ GATA 3 antibody or normal IgG as described in materials and methods. The purified DNA from immunoprecipitation and from the input samples were used to amplify a fragment (132 bp) between −2362 and −2230 (containing the GATA ‐2‐binding site) by PCR and the PCR products were examined by 2% agarose gel electrophoresis. ( C ) Immunoprecipitation assay of the interaction between GATA 3 and GATA 2 ( n = 3). After pF lag‐ GATA 3 (encoding Flag‐ GATA 3 protein) were cotransfected 293T cells with pc DNAGATA 2 (encoding mouse GATA 2 protein) and incubated for 24 hrs, 293T cells were lysed by RIPA buffer and the some of the cell lysate was used as input sample and the others were used for immunoprecipitation by using the anti‐Flag antibody or the normal IgG. Precipitated immune complexes were detected by western blot analysis by using antibody against GATA 2 or GATA 3. A representative result was shown. ( D ) Luciferase assay ( n = 3). The first group of HSC s were cotransfected with 0.8 μg of pPPAR γ1( GATA mut)Luc or 0.8 μg of pPPAR γ1(−2333)Luc plus 0.8 μg of pc DNAGATA 3 or 0.8 μg of the empty vector and then incubated for 24 hrs. The second group of HSC s were cotransfected with 0.8 μg of p3× GATAL uc or pGL 3‐promoter vector (control) plus 0.8 μg of pc DNAGATA 3 or empty vector and then incubated for 24 hrs. Luciferase assay was performed. * P

    Techniques Used: Binding Assay, Incubation, Competitive Binding Assay, Purification, Immunoprecipitation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Luciferase, Plasmid Preparation

    7) Product Images from "The Global Virulence Regulator PhcA Negatively Controls the Ralstonia solanacearum hrp Regulatory Cascade by Repressing Expression of the PrhIR Signaling Proteins ▿ Regulatory Cascade by Repressing Expression of the PrhIR Signaling Proteins ▿ †"

    Article Title: The Global Virulence Regulator PhcA Negatively Controls the Ralstonia solanacearum hrp Regulatory Cascade by Repressing Expression of the PrhIR Signaling Proteins ▿ Regulatory Cascade by Repressing Expression of the PrhIR Signaling Proteins ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01113-08

    Gel mobility shift assay for PhcA. (A) Biotinylated promoter DNA fragments are as follows: P xpsR1 , xpsR containing the PhcA binding region; P xpsR2 , xpsR lacking this region; P hrpB , hrpB ; P hrpG , hrpG ; P prhJ , prhJ ; P prhIR , prhIR ; P prhA , prhA . The length
    Figure Legend Snippet: Gel mobility shift assay for PhcA. (A) Biotinylated promoter DNA fragments are as follows: P xpsR1 , xpsR containing the PhcA binding region; P xpsR2 , xpsR lacking this region; P hrpB , hrpB ; P hrpG , hrpG ; P prhJ , prhJ ; P prhIR , prhIR ; P prhA , prhA . The length

    Techniques Used: Mobility Shift, Binding Assay

    Related Articles

    Incubation:

    Article Title: Continuous biomarker monitoring by particle mobility sensing with single molecule resolution
    Article Snippet: For each concentration, 400 µL of the solution was injected into the measurement chamber at a rate of 100 µL per min, after which the particle motion was recorded for 5 min in the absence of flow. .. Particle and surface functionalization for the thrombin system Ten µL of the streptavidin-coated magnetic particles (10 mg/mL, Dynabeads MyOne Streptavidin C1, 65001, Thermo Scientific) was incubated with 10 µL 120 bp dsDNA tether (with 5′ Digoxigenin and 5′ Biotin on either end) at a concentration of 2 nM in PBS buffer solution for 10 min on rotating fins (VWR, The Netherlands). .. Next, 10 µL PBS with 50 nM of the 29-mer aptamer (5′ Biotin-TTTTTTTTTTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′, Integrated DNA Technology) was adde d to the particle mixture and incubated on rotating fins for 30 min. Then 10 µL mPEG–Biotin (PG1-BN-1k, Nanocs) of 100 µM in PBS were incubated with the particle mixture for 5 min. After that, the particle mixture was washed three times with 500 µL PBS and reconstituted in 630 µL assay buffer (PBS with 1% BSA and 0.05% Tween 20 (Sigma Aldrich), filtered with a 0.22 µm filter and degassed in the desiccator) and incubated on the rotating fin for 30 min.

    Article Title: Continuous biomarker monitoring by particle mobility sensing with single molecule resolution
    Article Snippet: With its basis in affinity binding and single-molecule resolution, we envisage that the presented technology will enable biosensors for continuous biomarker monitoring with high sensitivity, specificity, and accuracy. .. Ten microliters of the streptavidin-coated magnetic particles (10 mg/mL, Dynabeads MyOne Streptavidin C1, 65001, Thermo Scientific) was incubated with 10 µL 120 bp dsDNA tether (with 5′ Digoxigenin and 5′ Biotin on either end) at a concentration of 2 nM in PBS buffer solution for 10 min on rotating fins (VWR, The Netherlands). .. Next, 10 µL PBS with 10 µM of capture molecule (11-nt oligo, 5′-TCACGGTACGA-3′ Biotin, Integrated DNA Technologies) was added to the particle mixture and incubated on rotating fins for 30 min. Then 10 µL mPEG-Biotin (PG1-BN-1k, Nanocs) of 100 µM in PBS were incubated with the particle mixture for 5 min. After that, the particle mixture was washed three times with 500 µL PBS, reconstituted in 630 µL PBS/BSA buffer (PBS with 1% BSA filtered with a 0.22 µm filter and degassed in a vacuum desiccator) and kept on the rotating fin for 30 min.

    Concentration Assay:

    Article Title: Continuous biomarker monitoring by particle mobility sensing with single molecule resolution
    Article Snippet: For each concentration, 400 µL of the solution was injected into the measurement chamber at a rate of 100 µL per min, after which the particle motion was recorded for 5 min in the absence of flow. .. Particle and surface functionalization for the thrombin system Ten µL of the streptavidin-coated magnetic particles (10 mg/mL, Dynabeads MyOne Streptavidin C1, 65001, Thermo Scientific) was incubated with 10 µL 120 bp dsDNA tether (with 5′ Digoxigenin and 5′ Biotin on either end) at a concentration of 2 nM in PBS buffer solution for 10 min on rotating fins (VWR, The Netherlands). .. Next, 10 µL PBS with 50 nM of the 29-mer aptamer (5′ Biotin-TTTTTTTTTTTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′, Integrated DNA Technology) was adde d to the particle mixture and incubated on rotating fins for 30 min. Then 10 µL mPEG–Biotin (PG1-BN-1k, Nanocs) of 100 µM in PBS were incubated with the particle mixture for 5 min. After that, the particle mixture was washed three times with 500 µL PBS and reconstituted in 630 µL assay buffer (PBS with 1% BSA and 0.05% Tween 20 (Sigma Aldrich), filtered with a 0.22 µm filter and degassed in the desiccator) and incubated on the rotating fin for 30 min.

    Article Title: Continuous biomarker monitoring by particle mobility sensing with single molecule resolution
    Article Snippet: With its basis in affinity binding and single-molecule resolution, we envisage that the presented technology will enable biosensors for continuous biomarker monitoring with high sensitivity, specificity, and accuracy. .. Ten microliters of the streptavidin-coated magnetic particles (10 mg/mL, Dynabeads MyOne Streptavidin C1, 65001, Thermo Scientific) was incubated with 10 µL 120 bp dsDNA tether (with 5′ Digoxigenin and 5′ Biotin on either end) at a concentration of 2 nM in PBS buffer solution for 10 min on rotating fins (VWR, The Netherlands). .. Next, 10 µL PBS with 10 µM of capture molecule (11-nt oligo, 5′-TCACGGTACGA-3′ Biotin, Integrated DNA Technologies) was added to the particle mixture and incubated on rotating fins for 30 min. Then 10 µL mPEG-Biotin (PG1-BN-1k, Nanocs) of 100 µM in PBS were incubated with the particle mixture for 5 min. After that, the particle mixture was washed three times with 500 µL PBS, reconstituted in 630 µL PBS/BSA buffer (PBS with 1% BSA filtered with a 0.22 µm filter and degassed in a vacuum desiccator) and kept on the rotating fin for 30 min.

    Binding Assay:

    Article Title: Simple and Efficient Room-Temperature Release of Biotinylated Nucleic Acids from Streptavidin and Its Application to Selective Molecular Detection
    Article Snippet: .. For bead capture measurements, 10 μ L of streptavidin-conjugated beads (l μ m diameter Dynabeads MyOne Streptavidin Cl beads, Invitrogen, Carlsbad, CA) were washed three times in 1× binding/washing buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl) before being resuspended in 10 μ L of 2X binding/washing buffer. .. Biotinylated dsDNA (10 μL) at a concentration of 50 nM was then added to the beads and agitated for 30 min.

    Article Title: Towards XNA molecular biology: Bacterial cell display as a robust and versatile platform for the engineering of low affinity ligands and enzymes
    Article Snippet: Labelled cells were added to streptavidin beads (5 µl, Dynabeads, MyOne C1, Thermo Fisher) in binding buffer (TN-DBT, 50 mM Tris•Cl pH 7.5, 10 mM NaCl, 1 mM DTT, 0.1% BSA, 0.01% Tween 20) and put through selection in a KingFisher™ Duo Purification System. .. Labelled cells were added to streptavidin beads (5 µl, Dynabeads, MyOne C1, Thermo Fisher) in binding buffer (TN-DBT, 50 mM Tris•Cl pH 7.5, 10 mM NaCl, 1 mM DTT, 0.1% BSA, 0.01% Tween 20) and put through selection in a KingFisher™ Duo Purification System. .. The protocol included a binding step (30 min, 37°C, medium shaking, collect beads 3x 5 s), 6 wash steps (5 min, medium shaking, collect beads 3x 1 s), and an elution step (into 50 µl PBS).

    Lysis:

    Article Title: Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia
    Article Snippet: Benzonase (1 μl, 250 U/μl; E1014; Sigma-Aldrich) was then added to each sample and incubated at 4°C for 1 hour to further digest chromatin. .. Biotinylated BSP probes [50 nmol conjugated to 20 μl of MyOne Streptavidin C1 Dynabeads (65002; Invitrogen) for at least an hour in 1× phosphate-buffered saline (PBS)] were washed with lysis buffer, and an equal bead volume was subsequently aliquoted between centrifuged cell lysates. .. The mixture was incubated for 2 hours at 4°C with gentle agitation (nutator) with or without competition from 30 nmol of BSP.

    Magnetic Beads:

    Article Title: Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells
    Article Snippet: Finally, RNA was reconstituted in 25-50 µL of RNase-free water. .. For removing of biotinylated 4sU-RNA, streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin C1 beads, ThermoFisher) were used according to the manufacturer’s manual. .. To avoid unfavorable secondary RNA structures that potentially impair the binding to the beads, the RNA was first denatured at 65°C for 10 min followed by rapid cooling on ice for 5 min. 200 µL of Dynabeads magnetic beads per sample was transferred to a new tube.

    Selection:

    Article Title: Towards XNA molecular biology: Bacterial cell display as a robust and versatile platform for the engineering of low affinity ligands and enzymes
    Article Snippet: Labelled cells were added to streptavidin beads (5 µl, Dynabeads, MyOne C1, Thermo Fisher) in binding buffer (TN-DBT, 50 mM Tris•Cl pH 7.5, 10 mM NaCl, 1 mM DTT, 0.1% BSA, 0.01% Tween 20) and put through selection in a KingFisher™ Duo Purification System. .. Labelled cells were added to streptavidin beads (5 µl, Dynabeads, MyOne C1, Thermo Fisher) in binding buffer (TN-DBT, 50 mM Tris•Cl pH 7.5, 10 mM NaCl, 1 mM DTT, 0.1% BSA, 0.01% Tween 20) and put through selection in a KingFisher™ Duo Purification System. .. The protocol included a binding step (30 min, 37°C, medium shaking, collect beads 3x 5 s), 6 wash steps (5 min, medium shaking, collect beads 3x 1 s), and an elution step (into 50 µl PBS).

    Purification:

    Article Title: Towards XNA molecular biology: Bacterial cell display as a robust and versatile platform for the engineering of low affinity ligands and enzymes
    Article Snippet: Labelled cells were added to streptavidin beads (5 µl, Dynabeads, MyOne C1, Thermo Fisher) in binding buffer (TN-DBT, 50 mM Tris•Cl pH 7.5, 10 mM NaCl, 1 mM DTT, 0.1% BSA, 0.01% Tween 20) and put through selection in a KingFisher™ Duo Purification System. .. Labelled cells were added to streptavidin beads (5 µl, Dynabeads, MyOne C1, Thermo Fisher) in binding buffer (TN-DBT, 50 mM Tris•Cl pH 7.5, 10 mM NaCl, 1 mM DTT, 0.1% BSA, 0.01% Tween 20) and put through selection in a KingFisher™ Duo Purification System. .. The protocol included a binding step (30 min, 37°C, medium shaking, collect beads 3x 5 s), 6 wash steps (5 min, medium shaking, collect beads 3x 1 s), and an elution step (into 50 µl PBS).

    Article Title: High-Spatial-Resolution Multi-Omics Atlas Sequencing of Mouse Embryos via Deterministic Barcoding in Tissue
    Article Snippet: Depending on the area of this region, the typical amount of buffer is 10 - 100 µL of Proteinase K lysis solution, which contains 2 mg/mL proteinase K (Thermo Fisher), 10 mM Tris (pH = 8.0), 200 mM NaCl, 50 mM EDTA and 2% SDS. .. The cDNAs in the lysate were purified using streptavidin beads (Dynabeads MyOne Streptavidin C1 beads, Thermo Fisher). .. The beads (40 µL) were first washed three times with 1X B & W buffer (Ref to manufacturer’s manual) with 0.05% Tween-20, and then stored in 100 µL of 2X B & W buffer (with 2 μL of SUPERase In Rnase Inhibitor).

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  • 97
    Thermo Fisher biotinylated dna fragments
    ME-Scan library preparation ( A–E ) and sequencing ( F–H ). ( A ) Genomic <t>DNA</t> (green) containing an Alu Yb8/9 (blue) in the forward orientation. The 5′ GC-rich region, the 3′ poly-A tail, and the Alu Yb8/9-specific primer site ( Alu BP2) are shown as darker segments. The target site duplications (TSD) are indicated by boxed black arrows. ( B ) DNA samples are fragmented, end-repaired, and an overhanging 3′ A is added. ( C ) Partially double-stranded oligonucleotide adapters (orange) with 9-bp indexes (darker segment) are ligated onto fragment ends. Indexed samples are then pooled. ( D ) Alu Yb8/9 element junctions are targeted by PCR using a <t>biotinylated</t> Alu Yb8/9-specific primer ( Alu BP2) and adapter primer PEP2. Biotinylated DNA molecules are then purified using streptavidin-coated paramagnetic beads. ( E ) Reamplification of the library with primers PEP1 and PEP2 (orange arrows). ( F–H ) The pooled junction library is then sequenced with a three-read design. Primers are shown as arrows, sequencing reads as dashed lines. The first sequencing read (50 nt) extends from Alu SPv2 ( F ) or Alu SPv3 ( G ). To skip the 5′ end of the Alu insertions, which would be identical over the entire flow cell and therefore difficult for Illumina's software to process, the first 30 cycles of nucleotide synthesis are carried out without collecting data (represented by a dashed gray ‘hop’). The second read proceeds for 57 bp from Alu SPIn1. The third read is generated using Illumina's standard second-end read primer and consists of a 9-bp index, a ‘T,’ and 36 bp of genomic sequence from a fragmentation site 50–300 bp upstream of the Alu insertion. Read sets are generated from multiple different fragments representing each Alu Yb8/9 insertion in the library ( H ). Each insertion is uniquely identified by its “ Alu Junction Position” (dashed line and large arrow in F and G ).
    Biotinylated Dna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher antibodies rabbit anti occludin
    Immunofluorescence of untreated Caco-2 cells. These cells were labelled with <t>occludin</t> (left), ZO-1 (middle) or E-cadherin (right) junctional proteins (FITC-green staining), actin (TRITC-red staining) and a DAPI nuclear counter-stain (blue). Top row illustrates
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    97
    Thermo Fisher cdna fragments
    aPKC is required for exocytosis of newly synthesized <t>nephrin.</t> ( A ) HeLa Tet-On Advanced cells were transiently transfected with nephrin <t>cDNA</t> and incubated for 48 h. After incubation, cells were treated with 20 µM of aPKC-PS or SC for 2 h, and then incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. ( B ) Quantification of the results in (A). ( C ) HeLa Tet-On Advanced cells were transiently transfected with nephrin and aPKC WT or KN cDNA, and incubated for 48 h. After incubation, the cells were incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. ( D ) Quantification of the results in (C). The values shown in B and D were normalized to those at the start of doxycycline treatment and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t -test.
    Cdna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ME-Scan library preparation ( A–E ) and sequencing ( F–H ). ( A ) Genomic DNA (green) containing an Alu Yb8/9 (blue) in the forward orientation. The 5′ GC-rich region, the 3′ poly-A tail, and the Alu Yb8/9-specific primer site ( Alu BP2) are shown as darker segments. The target site duplications (TSD) are indicated by boxed black arrows. ( B ) DNA samples are fragmented, end-repaired, and an overhanging 3′ A is added. ( C ) Partially double-stranded oligonucleotide adapters (orange) with 9-bp indexes (darker segment) are ligated onto fragment ends. Indexed samples are then pooled. ( D ) Alu Yb8/9 element junctions are targeted by PCR using a biotinylated Alu Yb8/9-specific primer ( Alu BP2) and adapter primer PEP2. Biotinylated DNA molecules are then purified using streptavidin-coated paramagnetic beads. ( E ) Reamplification of the library with primers PEP1 and PEP2 (orange arrows). ( F–H ) The pooled junction library is then sequenced with a three-read design. Primers are shown as arrows, sequencing reads as dashed lines. The first sequencing read (50 nt) extends from Alu SPv2 ( F ) or Alu SPv3 ( G ). To skip the 5′ end of the Alu insertions, which would be identical over the entire flow cell and therefore difficult for Illumina's software to process, the first 30 cycles of nucleotide synthesis are carried out without collecting data (represented by a dashed gray ‘hop’). The second read proceeds for 57 bp from Alu SPIn1. The third read is generated using Illumina's standard second-end read primer and consists of a 9-bp index, a ‘T,’ and 36 bp of genomic sequence from a fragmentation site 50–300 bp upstream of the Alu insertion. Read sets are generated from multiple different fragments representing each Alu Yb8/9 insertion in the library ( H ). Each insertion is uniquely identified by its “ Alu Junction Position” (dashed line and large arrow in F and G ).

    Journal: Genome Research

    Article Title: Mobile element scanning (ME-Scan) identifies thousands of novel Alu insertions in diverse human populations

    doi: 10.1101/gr.148973.112

    Figure Lengend Snippet: ME-Scan library preparation ( A–E ) and sequencing ( F–H ). ( A ) Genomic DNA (green) containing an Alu Yb8/9 (blue) in the forward orientation. The 5′ GC-rich region, the 3′ poly-A tail, and the Alu Yb8/9-specific primer site ( Alu BP2) are shown as darker segments. The target site duplications (TSD) are indicated by boxed black arrows. ( B ) DNA samples are fragmented, end-repaired, and an overhanging 3′ A is added. ( C ) Partially double-stranded oligonucleotide adapters (orange) with 9-bp indexes (darker segment) are ligated onto fragment ends. Indexed samples are then pooled. ( D ) Alu Yb8/9 element junctions are targeted by PCR using a biotinylated Alu Yb8/9-specific primer ( Alu BP2) and adapter primer PEP2. Biotinylated DNA molecules are then purified using streptavidin-coated paramagnetic beads. ( E ) Reamplification of the library with primers PEP1 and PEP2 (orange arrows). ( F–H ) The pooled junction library is then sequenced with a three-read design. Primers are shown as arrows, sequencing reads as dashed lines. The first sequencing read (50 nt) extends from Alu SPv2 ( F ) or Alu SPv3 ( G ). To skip the 5′ end of the Alu insertions, which would be identical over the entire flow cell and therefore difficult for Illumina's software to process, the first 30 cycles of nucleotide synthesis are carried out without collecting data (represented by a dashed gray ‘hop’). The second read proceeds for 57 bp from Alu SPIn1. The third read is generated using Illumina's standard second-end read primer and consists of a 9-bp index, a ‘T,’ and 36 bp of genomic sequence from a fragmentation site 50–300 bp upstream of the Alu insertion. Read sets are generated from multiple different fragments representing each Alu Yb8/9 insertion in the library ( H ). Each insertion is uniquely identified by its “ Alu Junction Position” (dashed line and large arrow in F and G ).

    Article Snippet: The size-selected DNA was incubated with streptavidin-coated paramagnetic beads to retain biotinylated DNA fragments per the manufacturer's protocol (Dynabeads MyOne Streptavidin C1, Life Technologies, Inc.).

    Techniques: Sequencing, Polymerase Chain Reaction, Purification, Flow Cytometry, Software, Generated

    Immunofluorescence of untreated Caco-2 cells. These cells were labelled with occludin (left), ZO-1 (middle) or E-cadherin (right) junctional proteins (FITC-green staining), actin (TRITC-red staining) and a DAPI nuclear counter-stain (blue). Top row illustrates

    Journal: Journal of Anatomy

    Article Title: Macrophages increase microparticle uptake by enterocyte-like Caco-2 cell monolayers

    doi: 10.1111/j.1469-7580.2010.01304.x

    Figure Lengend Snippet: Immunofluorescence of untreated Caco-2 cells. These cells were labelled with occludin (left), ZO-1 (middle) or E-cadherin (right) junctional proteins (FITC-green staining), actin (TRITC-red staining) and a DAPI nuclear counter-stain (blue). Top row illustrates

    Article Snippet: Samples were incubated with the primary antibodies rabbit anti-occludin (1 ng μL−1 ; Invitrogen 71–1500), rabbit anti-ZO-1 (25 ng μL−1 ; Invitrogen 61–7300) or mouse anti-E-cadherin (500 ng μL−1 ; Abcam ab1416), for 2 h. After washing, the cells were incubated for 1 h with biotinylated goat anti-rabbit IgG or biotinylated horse anti-mouse IgG (15 ng μL−1 ; Vector Laboratories) and then exposed to a mixture of fluorescein Avidin-D (20 ng μL−1 ; Vector Laboratories) and TRITC-labelled phalloidin-tetramethylrhodamine B for F-actin (20 ng μL−1 ; Sigma 77418), in PBS for 45 min.

    Techniques: Immunofluorescence, Staining

    aPKC is required for exocytosis of newly synthesized nephrin. ( A ) HeLa Tet-On Advanced cells were transiently transfected with nephrin cDNA and incubated for 48 h. After incubation, cells were treated with 20 µM of aPKC-PS or SC for 2 h, and then incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. ( B ) Quantification of the results in (A). ( C ) HeLa Tet-On Advanced cells were transiently transfected with nephrin and aPKC WT or KN cDNA, and incubated for 48 h. After incubation, the cells were incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. ( D ) Quantification of the results in (C). The values shown in B and D were normalized to those at the start of doxycycline treatment and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t -test.

    Journal: Journal of Biochemistry

    Article Title: aPKCλ maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface

    doi: 10.1093/jb/mvu022

    Figure Lengend Snippet: aPKC is required for exocytosis of newly synthesized nephrin. ( A ) HeLa Tet-On Advanced cells were transiently transfected with nephrin cDNA and incubated for 48 h. After incubation, cells were treated with 20 µM of aPKC-PS or SC for 2 h, and then incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. ( B ) Quantification of the results in (A). ( C ) HeLa Tet-On Advanced cells were transiently transfected with nephrin and aPKC WT or KN cDNA, and incubated for 48 h. After incubation, the cells were incubated with 100 ng/ml doxycycline for the indicated times to induce the expression of nephrin. After doxycycline treatment, the cells were subjected to the cell-surface biotinylation assay. ( D ) Quantification of the results in (C). The values shown in B and D were normalized to those at the start of doxycycline treatment and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t -test.

    Article Snippet: Expression constructs and siRNA Human nephrin and podocin cDNA was described previously ( ). cDNA fragments of human nephrin were amplified by PCR, and subcloned into pcDNA5-FRT-TO (Life Technologies) or pTRET-FRT-Hyg-TetOn vectors.

    Techniques: Synthesized, Transfection, Incubation, Expressing, Cell Surface Biotinylation Assay, Two Tailed Test

    aPKC is required for the cell-surface localization of SD components, including nephrin. ( A ) Isolated rat glomeruli were treated with 10 µM aPKC pseudosubstrate (PS) or SC for 30 min at 37°C in HBSS(+), then subjected to the cell-surface biotinylation assay. ( B ) Quantification of the results in (A). ( C ) HCT116-nephrin cells were treated with 20 µM of aPKC-PS or SC for 2 h at 37°C and subjected to the cell-surface biotinylation assay. ( D ) Quantification of the results in (C). ( E ) HCT116-nephrin cells were transiently transfected with aPKC WT or KN cDNA and incubated for 48 h and then subjected to the cell-surface biotinylation assay. ( F ) Quantification of the results in (E). ( G ) HCT116-nephrin cells were transiently transfected with aPKCλ/ζ siRNA and incubated for 70 h. Both isotypes of aPKC are expressed in HCT116 cells (data not shown). After incubation, the cells were subjected to the cell-surface biotinylation assay. ( H ) Quantification of the results in (G). The values shown in B, D, F and H were normalized to the appropriate control and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t -test.

    Journal: Journal of Biochemistry

    Article Title: aPKCλ maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface

    doi: 10.1093/jb/mvu022

    Figure Lengend Snippet: aPKC is required for the cell-surface localization of SD components, including nephrin. ( A ) Isolated rat glomeruli were treated with 10 µM aPKC pseudosubstrate (PS) or SC for 30 min at 37°C in HBSS(+), then subjected to the cell-surface biotinylation assay. ( B ) Quantification of the results in (A). ( C ) HCT116-nephrin cells were treated with 20 µM of aPKC-PS or SC for 2 h at 37°C and subjected to the cell-surface biotinylation assay. ( D ) Quantification of the results in (C). ( E ) HCT116-nephrin cells were transiently transfected with aPKC WT or KN cDNA and incubated for 48 h and then subjected to the cell-surface biotinylation assay. ( F ) Quantification of the results in (E). ( G ) HCT116-nephrin cells were transiently transfected with aPKCλ/ζ siRNA and incubated for 70 h. Both isotypes of aPKC are expressed in HCT116 cells (data not shown). After incubation, the cells were subjected to the cell-surface biotinylation assay. ( H ) Quantification of the results in (G). The values shown in B, D, F and H were normalized to the appropriate control and are the mean ± SD of three independent experiments. The P values were determined by two-tailed Student’s t -test.

    Article Snippet: Expression constructs and siRNA Human nephrin and podocin cDNA was described previously ( ). cDNA fragments of human nephrin were amplified by PCR, and subcloned into pcDNA5-FRT-TO (Life Technologies) or pTRET-FRT-Hyg-TetOn vectors.

    Techniques: Isolation, Cell Surface Biotinylation Assay, Transfection, Incubation, Two Tailed Test

    aPKC does not suppress the endocytosis of nephrin. ( A ) HCT116-nephrin cells were treated with 20 µM aPKC-PS or SC for 2 h at 37°C and then subjected to the endocytosis assay as in Fig. 1 D. ( B ) Quantification of the results in (A). ( C ) HCT116-nephrin cells were transiently transfected with aPKC WT or KN cDNA and incubate for 48 h and then subjected to the endocytosis assay. ( D ) Quantification of the results in (C). The amount of endocytosed nephrin shown in B and D was expressed as the percentage of those at the start of labelling and are the mean ± SD of three independent experiments. ( E ) HCT116-nephrin cells were treated with 20 µM aPKC PS with or without 10 µM chlorpromazine or 10 mM MβCD for 30 min at 37°C and then subjected to the cell-surface biotinylation assay.

    Journal: Journal of Biochemistry

    Article Title: aPKCλ maintains the integrity of the glomerular slit diaphragm through trafficking of nephrin to the cell surface

    doi: 10.1093/jb/mvu022

    Figure Lengend Snippet: aPKC does not suppress the endocytosis of nephrin. ( A ) HCT116-nephrin cells were treated with 20 µM aPKC-PS or SC for 2 h at 37°C and then subjected to the endocytosis assay as in Fig. 1 D. ( B ) Quantification of the results in (A). ( C ) HCT116-nephrin cells were transiently transfected with aPKC WT or KN cDNA and incubate for 48 h and then subjected to the endocytosis assay. ( D ) Quantification of the results in (C). The amount of endocytosed nephrin shown in B and D was expressed as the percentage of those at the start of labelling and are the mean ± SD of three independent experiments. ( E ) HCT116-nephrin cells were treated with 20 µM aPKC PS with or without 10 µM chlorpromazine or 10 mM MβCD for 30 min at 37°C and then subjected to the cell-surface biotinylation assay.

    Article Snippet: Expression constructs and siRNA Human nephrin and podocin cDNA was described previously ( ). cDNA fragments of human nephrin were amplified by PCR, and subcloned into pcDNA5-FRT-TO (Life Technologies) or pTRET-FRT-Hyg-TetOn vectors.

    Techniques: Endocytosis Assay, Transfection, Cell Surface Biotinylation Assay