biotinylated dna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher biotinylated dna
    Poly(ADP-ribosyl)ation alters protein binding to the DS element. (A) Raji nuclear extracts were subjected to affinity chromatography with <t>biotinylated</t> DS element or control BKS <t>DNA.</t> Purified complexes coupled with beads were then incubated with buffer
    Biotinylated Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 25 article reviews
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    Images

    1) Product Images from "Regulation of Epstein-Barr Virus OriP Replication by Poly(ADP-Ribose) Polymerase 1 ▿"

    Article Title: Regulation of Epstein-Barr Virus OriP Replication by Poly(ADP-Ribose) Polymerase 1 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02333-09

    Poly(ADP-ribosyl)ation alters protein binding to the DS element. (A) Raji nuclear extracts were subjected to affinity chromatography with biotinylated DS element or control BKS DNA. Purified complexes coupled with beads were then incubated with buffer
    Figure Legend Snippet: Poly(ADP-ribosyl)ation alters protein binding to the DS element. (A) Raji nuclear extracts were subjected to affinity chromatography with biotinylated DS element or control BKS DNA. Purified complexes coupled with beads were then incubated with buffer

    Techniques Used: Protein Binding, Affinity Chromatography, Purification, Incubation

    2) Product Images from "The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin"

    Article Title: The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01981-17

    BrlR activates the expression of PA1874 and PA1875 and binds to the promoter region of PA1874-77. (A) Fold change in the abundance of the PA1874 and PA1875 transcripts in biofilms formed by the Δ brlR mutant and PAO1/pJN- brlR relative to the abundance in wild-type biofilms, as determined using qRT-PCR. The mreB housekeeping gene was used for the normalization of transcript abundance. (B) Fold change in the abundance of the PA1874, PA1875, and PA1877 transcripts in the Δ brlR mutant and PAO1/pMJT- brlR grown planktonically to exponential phase relative to that in wild-type strain PAO1. (C) Fold enrichment of the promoter DNA of PA1874, brlR , and pscEF in samples with BrlR-V5/His 6 submitted to ChIP compared to control samples (for which ChIP was carried out in the presence of untagged BrlR), as determined by qPCR. P pscEF was used as a negative control, while enrichment of the brlR promoter region (P brlR ) was used as a positive control. (D) Streptavidin (SA) magnetic bead binding assay demonstrating tagged BrlR binding to the biotinylated PA1874 promoter. BrlR binding to the PA1874 promoter was outcompeted using nonbiotinylated promoter DNA (P PA1874 -NB).
    Figure Legend Snippet: BrlR activates the expression of PA1874 and PA1875 and binds to the promoter region of PA1874-77. (A) Fold change in the abundance of the PA1874 and PA1875 transcripts in biofilms formed by the Δ brlR mutant and PAO1/pJN- brlR relative to the abundance in wild-type biofilms, as determined using qRT-PCR. The mreB housekeeping gene was used for the normalization of transcript abundance. (B) Fold change in the abundance of the PA1874, PA1875, and PA1877 transcripts in the Δ brlR mutant and PAO1/pMJT- brlR grown planktonically to exponential phase relative to that in wild-type strain PAO1. (C) Fold enrichment of the promoter DNA of PA1874, brlR , and pscEF in samples with BrlR-V5/His 6 submitted to ChIP compared to control samples (for which ChIP was carried out in the presence of untagged BrlR), as determined by qPCR. P pscEF was used as a negative control, while enrichment of the brlR promoter region (P brlR ) was used as a positive control. (D) Streptavidin (SA) magnetic bead binding assay demonstrating tagged BrlR binding to the biotinylated PA1874 promoter. BrlR binding to the PA1874 promoter was outcompeted using nonbiotinylated promoter DNA (P PA1874 -NB).

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Binding Assay

    3) Product Images from "The long zinc finger domain of PRDM9 forms a highly stable and long-lived complex with its DNA recognition sequence"

    Article Title: The long zinc finger domain of PRDM9 forms a highly stable and long-lived complex with its DNA recognition sequence

    Journal: Chromosome Research

    doi: 10.1007/s10577-017-9552-1

    PRDM9-DNA-complex stability assessed with an EMSA competition assay. Different concentrations of the PRDM9 Cst -ZnF domain (2284 or 150 nM) were incubated with 10 nM of a biotinylated 75 bp DNA (hot Hlx1 B6 ) and a 100-fold excess of 39 bp competitor DNA without biotin (cold Hlx1 B6 ). The competitor was added to the hot Hlx1 B6 at different time points. Lanes 2 and 7 show the incubation of PRDM9 only with hot Hlx1 B6 . In comparison, lanes 3 and 8 represent the simultaneous incubation of PRDM9 with hot and cold Hlx1 B6 and lanes 4 + 5 and 9 + 10 show the pre-incubation of PRDM9 with hot Hlx1 B6 for 1 h before adding the excess cold Hlx1 B6 to the reaction, which was then stopped either after 1 or ∼14 h. The average percentage of hot Hlx1 B6 in the PRDM9-DNA complex (% fraction bound) was estimated as the ratio of pixel intensities of the shifted band to the sum of free and bound hot Hlx1 B6 . Error bars represent the standard deviation of two independent experiments. The length difference between hot and cold Hlx1 B6 was important for the proper blotting of the free hot Hlx1 B6 used to quantitate the complex (fraction bound [%])
    Figure Legend Snippet: PRDM9-DNA-complex stability assessed with an EMSA competition assay. Different concentrations of the PRDM9 Cst -ZnF domain (2284 or 150 nM) were incubated with 10 nM of a biotinylated 75 bp DNA (hot Hlx1 B6 ) and a 100-fold excess of 39 bp competitor DNA without biotin (cold Hlx1 B6 ). The competitor was added to the hot Hlx1 B6 at different time points. Lanes 2 and 7 show the incubation of PRDM9 only with hot Hlx1 B6 . In comparison, lanes 3 and 8 represent the simultaneous incubation of PRDM9 with hot and cold Hlx1 B6 and lanes 4 + 5 and 9 + 10 show the pre-incubation of PRDM9 with hot Hlx1 B6 for 1 h before adding the excess cold Hlx1 B6 to the reaction, which was then stopped either after 1 or ∼14 h. The average percentage of hot Hlx1 B6 in the PRDM9-DNA complex (% fraction bound) was estimated as the ratio of pixel intensities of the shifted band to the sum of free and bound hot Hlx1 B6 . Error bars represent the standard deviation of two independent experiments. The length difference between hot and cold Hlx1 B6 was important for the proper blotting of the free hot Hlx1 B6 used to quantitate the complex (fraction bound [%])

    Techniques Used: Competitive Binding Assay, Incubation, Standard Deviation

    Binding affinity of PRDM9 to DNA in solution assessed with an EMSA titration assay. a Increasing concentrations of PRDM9 Cst -ZnF (0.16–4300 nM) were incubated with 3 nM of different biotinylated target DNAs ( Hlx1 B6 _75bp shown in black , Hlx1 Cst _76bp in red , and usDNA_75bp in blue , respectively) for ∼90 h, containing increasing concentrations of polydIdC (0.00035–9.39 ng/μl) and 0.03% sarcosyl ( N -lauroylsarcosine). Shown is a representative EMSA for Hlx1 B6 ( top ), Hlx1 Cst ( middle ), and usDNA ( bottom ) of triplicate ( Hlx1 ) or duplicate (usDNA) measurements. The average percent of the PRDM9-DNA complexes (% fraction bound) was calculated as the ratio of pixel intensities of the shifted band to the sum of free and bound DNA. b The average fraction bound of two (usDNA) or three ( Hlx1 B6 and Hlx1 Cst ) independent experiments was plotted against the PRDM9 concentration in a semi-logarithmic graph and the equilibrium dissociation constant ( K D ) was derived using a function that describes receptor-ligand binding in solution in dependence of the concentration of the labeled compound (see Supplementary_Methods). The error bars represent the standard deviation of two or three independent experiments. c Shown is one strand of the DNA sequences tested: Hlx1 B6 , Hlx1 Cst , and usDNA in black , red , and blue , respectively. All DNA sequences are shown in 5′–3′ direction. The specific target site of the PRDM9 Cst -ZnF array in the Hlx1 hotspots is highlighted in yellow . The polymorphisms between Hlx1 B6 and Hlx1 Cst are highlighted as bold , underlined letters
    Figure Legend Snippet: Binding affinity of PRDM9 to DNA in solution assessed with an EMSA titration assay. a Increasing concentrations of PRDM9 Cst -ZnF (0.16–4300 nM) were incubated with 3 nM of different biotinylated target DNAs ( Hlx1 B6 _75bp shown in black , Hlx1 Cst _76bp in red , and usDNA_75bp in blue , respectively) for ∼90 h, containing increasing concentrations of polydIdC (0.00035–9.39 ng/μl) and 0.03% sarcosyl ( N -lauroylsarcosine). Shown is a representative EMSA for Hlx1 B6 ( top ), Hlx1 Cst ( middle ), and usDNA ( bottom ) of triplicate ( Hlx1 ) or duplicate (usDNA) measurements. The average percent of the PRDM9-DNA complexes (% fraction bound) was calculated as the ratio of pixel intensities of the shifted band to the sum of free and bound DNA. b The average fraction bound of two (usDNA) or three ( Hlx1 B6 and Hlx1 Cst ) independent experiments was plotted against the PRDM9 concentration in a semi-logarithmic graph and the equilibrium dissociation constant ( K D ) was derived using a function that describes receptor-ligand binding in solution in dependence of the concentration of the labeled compound (see Supplementary_Methods). The error bars represent the standard deviation of two or three independent experiments. c Shown is one strand of the DNA sequences tested: Hlx1 B6 , Hlx1 Cst , and usDNA in black , red , and blue , respectively. All DNA sequences are shown in 5′–3′ direction. The specific target site of the PRDM9 Cst -ZnF array in the Hlx1 hotspots is highlighted in yellow . The polymorphisms between Hlx1 B6 and Hlx1 Cst are highlighted as bold , underlined letters

    Techniques Used: Binding Assay, Titration, Incubation, Concentration Assay, Derivative Assay, Ligand Binding Assay, Labeling, Standard Deviation

    PRDM9 binding to different lengths of DNA. a In this competition EMSA, 250 nM PRDM9 Cst -ZnF (YFP tagged) was incubated simultaneously with hot (biotinylated) reference DNA (75 bp Hlx1 B6 ) and different ratios of excess cold Hlx1 B6 for 1 h (a time frame that showed unchanged complex amounts; Supplementary_Fig_ S4 ). We used for each experiment different lengths of cold DNA (75 bp , 39 bp , 34 bp , 31 bp , 28 bp-d , and 28 bp-u sequences are shown in Supplementary_Table_ S1 , section C ) representing truncations of the 75 bp Hlx1 B6 sequence. Shown are representative EMSAs for the cold competitors with 31 and 28 bp. The measurements were performed in triplicates for each cold competitor. b Intensities of the shifted bands are calculated relative to the reference PRDM9 Cst - Hlx1 B6 complex without the addition of cold DNA ( lane 2 ) and are plotted as relative intensities against the concentration of the cold competitor in a semi-logarithmic graph. 28 bp-d and 28 bp-u indicate 28 base-pair long fragments with truncations of three bases from the target binding site at either the downstream (3′ end) or the upstream (5′ end) end, respectively
    Figure Legend Snippet: PRDM9 binding to different lengths of DNA. a In this competition EMSA, 250 nM PRDM9 Cst -ZnF (YFP tagged) was incubated simultaneously with hot (biotinylated) reference DNA (75 bp Hlx1 B6 ) and different ratios of excess cold Hlx1 B6 for 1 h (a time frame that showed unchanged complex amounts; Supplementary_Fig_ S4 ). We used for each experiment different lengths of cold DNA (75 bp , 39 bp , 34 bp , 31 bp , 28 bp-d , and 28 bp-u sequences are shown in Supplementary_Table_ S1 , section C ) representing truncations of the 75 bp Hlx1 B6 sequence. Shown are representative EMSAs for the cold competitors with 31 and 28 bp. The measurements were performed in triplicates for each cold competitor. b Intensities of the shifted bands are calculated relative to the reference PRDM9 Cst - Hlx1 B6 complex without the addition of cold DNA ( lane 2 ) and are plotted as relative intensities against the concentration of the cold competitor in a semi-logarithmic graph. 28 bp-d and 28 bp-u indicate 28 base-pair long fragments with truncations of three bases from the target binding site at either the downstream (3′ end) or the upstream (5′ end) end, respectively

    Techniques Used: Binding Assay, Incubation, Sequencing, Concentration Assay

    4) Product Images from "Divide and conquer: The Pseudomonas aeruginosa two-component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits"

    Article Title: Divide and conquer: The Pseudomonas aeruginosa two-component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits

    Journal: Environmental microbiology

    doi: 10.1111/1462-2920.13719

    Multicopy expression of sagS -HmsP-HisKA restores BrlR levels and enables BrlR-DNA binding to wild-type levels (A) Detection of BrlR by immunoblot analysis. Total cell extracts (TCE) obtained from P. aeruginosa wild-type and Δ sagS mutant biofilms (3-day old) expressing a chromosomally located V5/His 6 -tagged BrlR under the control of its own promoter (P brlR - brlR -V5/His 6 ) were probed for the presence of BrlR by immunoblot analysis (IB) using anti-V5 antibodies (anti-V5). A total of 15µg total cell extract was loaded. The corresponding SDS/PAGE gel image obtained post-transfer demonstrates equal loading. (B) BrlR-DNA binding using streptavidin magnetic bead binding assays and cell extracts obtained from PAO1 and Δ sagS mutant biofilms (3-day old) overexpressing V5/His 6 -tagged BrlR and sagS or SagS domain constructs. Binding assays were carried out using a total of 5pmol the BrlR-V5/His 6 protein obtained from the strains indicated, and 1 pmol biotinylated P brlR . Non-biotinylated P brlR (P brlR -NB) was used as specific competitor DNA in 20-fold excess. BrlR binding to P brlR ). BrlR protein production refers to BrlR abundance based on band intensity obtained following immunoblot analysis using total cell extracts. BrlR-P blR binding refers to BrlR abundance obtained following analysis of the streptavidin magnetic bead binding assays. Experiments were carried out in triplicate and representative images are shown. Error bars denote standard deviation. *, significantly different from the values for P. aeruginosa PAO1 ( p ≤ 0.01), as determined by ANOVA and SigmaStat.
    Figure Legend Snippet: Multicopy expression of sagS -HmsP-HisKA restores BrlR levels and enables BrlR-DNA binding to wild-type levels (A) Detection of BrlR by immunoblot analysis. Total cell extracts (TCE) obtained from P. aeruginosa wild-type and Δ sagS mutant biofilms (3-day old) expressing a chromosomally located V5/His 6 -tagged BrlR under the control of its own promoter (P brlR - brlR -V5/His 6 ) were probed for the presence of BrlR by immunoblot analysis (IB) using anti-V5 antibodies (anti-V5). A total of 15µg total cell extract was loaded. The corresponding SDS/PAGE gel image obtained post-transfer demonstrates equal loading. (B) BrlR-DNA binding using streptavidin magnetic bead binding assays and cell extracts obtained from PAO1 and Δ sagS mutant biofilms (3-day old) overexpressing V5/His 6 -tagged BrlR and sagS or SagS domain constructs. Binding assays were carried out using a total of 5pmol the BrlR-V5/His 6 protein obtained from the strains indicated, and 1 pmol biotinylated P brlR . Non-biotinylated P brlR (P brlR -NB) was used as specific competitor DNA in 20-fold excess. BrlR binding to P brlR ). BrlR protein production refers to BrlR abundance based on band intensity obtained following immunoblot analysis using total cell extracts. BrlR-P blR binding refers to BrlR abundance obtained following analysis of the streptavidin magnetic bead binding assays. Experiments were carried out in triplicate and representative images are shown. Error bars denote standard deviation. *, significantly different from the values for P. aeruginosa PAO1 ( p ≤ 0.01), as determined by ANOVA and SigmaStat.

    Techniques Used: Expressing, Binding Assay, Mutagenesis, SDS Page, Construct, Standard Deviation

    5) Product Images from "Menin enhances c-Myc-mediated transcription to promote cancer progression"

    Article Title: Menin enhances c-Myc-mediated transcription to promote cancer progression

    Journal: Nature Communications

    doi: 10.1038/ncomms15278

    Menin binds to E-box through interacting with MYC. ( a , b ) HEK293T cells were transfected with Flag-Menin or HA-MYC or both vectors. IP was performed with anti-Flag ( a ) or anti-HA ( b ), followed by immunoblot analysis. M: protein marker. ( c ) GST pull-down assay was performed with GST or GST fused MYC protein and His-Menin protein. ( d ) Endogenous MYC and Menin interaction. IP of endogenous Menin, MYC and MAX proteins from 293T cells lysed in high-salt buffer. IP was performed with anti-IgG or anti-Mein antibody. ( e ) GST or GST fused MYC truncated proteins TAD, CP and bHLHZ were purified (on the bottom) and used for GST pull-down assay with His-Menin. FL, full length. ( f ) GST pull-down assay was performed with GST or GST fused MAX protein and His-MYC (left panel) or His-Menin (right panel). ( g ) EMSA assays of purified MAX/MAX, MYC/MAX or Menin binding to biotinylated canonical CACGTG E-box sequences following competition with decreasing amounts (20-, 10-, 5-, 2- and 1-fold excess) of unlabelled competitor sequences. The higher bands reflect the amounts of labelled E-box bound by MYC/MAX or MAX/MAX and the lower band reflects the amount of unbound E-box DNA. Lane 1: no proteins. Lane 2: MAX/MAX with no competitor DNA. Lane 3: MYC/MAX with no competitor DNA. Lanes 4–8: the effect of adding indicated amounts of competitor DNA fragments containing the canonical E-box. Lane 9: Menin with no competitor DNA. Bottom: free biotinylated DNA. ( h ) EMSA assay analysing the binding of MAX/MAX, MYC/MAX or MYC/MAX/Menin to biotinylated canonical CACGTG E-box sequences in the presence or absence of anti-Menin antibody.
    Figure Legend Snippet: Menin binds to E-box through interacting with MYC. ( a , b ) HEK293T cells were transfected with Flag-Menin or HA-MYC or both vectors. IP was performed with anti-Flag ( a ) or anti-HA ( b ), followed by immunoblot analysis. M: protein marker. ( c ) GST pull-down assay was performed with GST or GST fused MYC protein and His-Menin protein. ( d ) Endogenous MYC and Menin interaction. IP of endogenous Menin, MYC and MAX proteins from 293T cells lysed in high-salt buffer. IP was performed with anti-IgG or anti-Mein antibody. ( e ) GST or GST fused MYC truncated proteins TAD, CP and bHLHZ were purified (on the bottom) and used for GST pull-down assay with His-Menin. FL, full length. ( f ) GST pull-down assay was performed with GST or GST fused MAX protein and His-MYC (left panel) or His-Menin (right panel). ( g ) EMSA assays of purified MAX/MAX, MYC/MAX or Menin binding to biotinylated canonical CACGTG E-box sequences following competition with decreasing amounts (20-, 10-, 5-, 2- and 1-fold excess) of unlabelled competitor sequences. The higher bands reflect the amounts of labelled E-box bound by MYC/MAX or MAX/MAX and the lower band reflects the amount of unbound E-box DNA. Lane 1: no proteins. Lane 2: MAX/MAX with no competitor DNA. Lane 3: MYC/MAX with no competitor DNA. Lanes 4–8: the effect of adding indicated amounts of competitor DNA fragments containing the canonical E-box. Lane 9: Menin with no competitor DNA. Bottom: free biotinylated DNA. ( h ) EMSA assay analysing the binding of MAX/MAX, MYC/MAX or MYC/MAX/Menin to biotinylated canonical CACGTG E-box sequences in the presence or absence of anti-Menin antibody.

    Techniques Used: Transfection, Marker, Pull Down Assay, Purification, Binding Assay

    6) Product Images from "Mobile element scanning (ME-Scan) by targeted high-throughput sequencing"

    Article Title: Mobile element scanning (ME-Scan) by targeted high-throughput sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-410

    Mobile Element Scanning (ME-Scan) Library Preparation and Sequencing Protocol . (A) dsDNA genomic DNA is extracted and then fragmented by sonication. An AluYb8/9 element is depicted (black rectangle: Alu element; gray box: Poly-A tail of the Alu ; TSD: t arget s ite d uplication). Some fragments (darker) will contain most or all of the element along with some upstream genomic sequence. (B) Fragment ends are repaired, 3'A overhangs are added, and oligonucleotide adapters (pink) carrying sample-specific indexes (blue) are ligated onto the ends. (C) Multiple indexed libraries are pooled for subsequent processing. (D) A limited number of PCR cycles are performed using a biotinylated AluYb8/9 -specific PCR primer (ALUBP2) and a primer (PEP2) that anneals to the adapters. PCR products in the 650-700 bp size range are selected using gel electrophoresis. (E) The biotinylated strands are purified away from other products using streptavidin-coated paramagnetic beads. (F) The biotinylated strands are amplified by PCR with primers matching the adapter sequences. The resulting product is checked using an Agilent Bioanalyzer DNA 1000 assay (electropherogram and gel-like image shown.) (G) Paired-end, 2x36-bp sequencing is carried out on the AluYb8/9 -specific pooled fragment library using a custom Alu -specific primer (ALUSPv2) for the first ( Alu junction) read and the standard adapter-specific primer (PESP2) for the second (genomic flank) read. The junction read begins inside the Alu element, yielding 16 bp of Alu sequence followed by 20 bp of genomic flank sequence. The flank read contains the 5-bp index and the 'T' added during sample preparation, followed by 30 bp of genomic sequence. Multiple read pairs are depicted, corresponding to different fragments carrying the same AluYb8/9 insertion (generic fragment diagrammed at bottom.)
    Figure Legend Snippet: Mobile Element Scanning (ME-Scan) Library Preparation and Sequencing Protocol . (A) dsDNA genomic DNA is extracted and then fragmented by sonication. An AluYb8/9 element is depicted (black rectangle: Alu element; gray box: Poly-A tail of the Alu ; TSD: t arget s ite d uplication). Some fragments (darker) will contain most or all of the element along with some upstream genomic sequence. (B) Fragment ends are repaired, 3'A overhangs are added, and oligonucleotide adapters (pink) carrying sample-specific indexes (blue) are ligated onto the ends. (C) Multiple indexed libraries are pooled for subsequent processing. (D) A limited number of PCR cycles are performed using a biotinylated AluYb8/9 -specific PCR primer (ALUBP2) and a primer (PEP2) that anneals to the adapters. PCR products in the 650-700 bp size range are selected using gel electrophoresis. (E) The biotinylated strands are purified away from other products using streptavidin-coated paramagnetic beads. (F) The biotinylated strands are amplified by PCR with primers matching the adapter sequences. The resulting product is checked using an Agilent Bioanalyzer DNA 1000 assay (electropherogram and gel-like image shown.) (G) Paired-end, 2x36-bp sequencing is carried out on the AluYb8/9 -specific pooled fragment library using a custom Alu -specific primer (ALUSPv2) for the first ( Alu junction) read and the standard adapter-specific primer (PESP2) for the second (genomic flank) read. The junction read begins inside the Alu element, yielding 16 bp of Alu sequence followed by 20 bp of genomic flank sequence. The flank read contains the 5-bp index and the 'T' added during sample preparation, followed by 30 bp of genomic sequence. Multiple read pairs are depicted, corresponding to different fragments carrying the same AluYb8/9 insertion (generic fragment diagrammed at bottom.)

    Techniques Used: Sequencing, Sonication, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Purification, Amplification, Sample Prep

    7) Product Images from "A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention"

    Article Title: A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention

    Journal: Journal of Virology

    doi: 10.1128/JVI.02143-18

    PcG proteins bind to replicating HCMV genomes. (A) HFF cells were infected with AD169 at an MOI of 1.5. At 72 hpi, newly synthesized DNA was labeled with EdU (10 μM) for 4 h, and Alexa 488 azide was conjugated by click chemistry. The viral protein pUL44 was visualized by antibody staining. The samples were analyzed by confocal microscopy. (B) Graph representing the percentage of cells positive for EdU only or for both EdU and pUL44 (mock, n = 150; infected, n = 150). (C) HFF cells were infected with AD169 at an MOI of 1.5 and used for accelerated native isolation of proteins on nascent DNA (aniPOND). At 72 hpi, cells were labeled for 4 h and biotinylated by click chemistry for affinity purification. No click reaction served as the negative control.
    Figure Legend Snippet: PcG proteins bind to replicating HCMV genomes. (A) HFF cells were infected with AD169 at an MOI of 1.5. At 72 hpi, newly synthesized DNA was labeled with EdU (10 μM) for 4 h, and Alexa 488 azide was conjugated by click chemistry. The viral protein pUL44 was visualized by antibody staining. The samples were analyzed by confocal microscopy. (B) Graph representing the percentage of cells positive for EdU only or for both EdU and pUL44 (mock, n = 150; infected, n = 150). (C) HFF cells were infected with AD169 at an MOI of 1.5 and used for accelerated native isolation of proteins on nascent DNA (aniPOND). At 72 hpi, cells were labeled for 4 h and biotinylated by click chemistry for affinity purification. No click reaction served as the negative control.

    Techniques Used: Infection, Synthesized, Labeling, Staining, Confocal Microscopy, Isolation, Affinity Purification, Negative Control

    8) Product Images from "GLUCOCORTICOIDS INHIBIT NOTCH TARGET GENE EXPRESSION IN OSTEOBLASTS"

    Article Title: GLUCOCORTICOIDS INHIBIT NOTCH TARGET GENE EXPRESSION IN OSTEOBLASTS

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.26798

    Cortisol has a modest impact on RBPJ/DNA interactions. Primary osteoblast-enriched cells were seeded on BSA or DLL1, confluent cultures maintained for 24 h in the absence of serum and then exposed to vehicle or cortisol 1 μM for 6 h. Nuclear proteins were extracted and binding reactions carried out with a biotinylated oligonucleotide containing an RBPJ consensus sequence from the EBNA2 promoter. Competition of binding reactions was performed in the presence of unlabeled oligonucleotides containing homologous RBPJ consensus sequences in 200-fold excess. DNA-nuclear protein complexes were resolved by gel electrophoresis, transferred to a nylon membrane, cross-linked with UV-light, exposed to a streptavidin-horseradish peroxidase conjugate and visualized by chemiluminescence. The arrows indicate the position of the DNA/protein complexes.
    Figure Legend Snippet: Cortisol has a modest impact on RBPJ/DNA interactions. Primary osteoblast-enriched cells were seeded on BSA or DLL1, confluent cultures maintained for 24 h in the absence of serum and then exposed to vehicle or cortisol 1 μM for 6 h. Nuclear proteins were extracted and binding reactions carried out with a biotinylated oligonucleotide containing an RBPJ consensus sequence from the EBNA2 promoter. Competition of binding reactions was performed in the presence of unlabeled oligonucleotides containing homologous RBPJ consensus sequences in 200-fold excess. DNA-nuclear protein complexes were resolved by gel electrophoresis, transferred to a nylon membrane, cross-linked with UV-light, exposed to a streptavidin-horseradish peroxidase conjugate and visualized by chemiluminescence. The arrows indicate the position of the DNA/protein complexes.

    Techniques Used: Binding Assay, Sequencing, Nucleic Acid Electrophoresis

    9) Product Images from "Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans"

    Article Title: Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0641-1

    Detection of the K222 provirus in the genome of human cell lines by slot blot analysis. The DNA of human cell lines that were found to have or lack the 5′ end of K111 by PCR, and presumably contain the truncated K222 provirus, were screened for K111 and K222 by slot blot analyses. (A) Generation of K111 and K222-specific biotinylated probes. Probes were generated by PCR incorporation of biotin-labeled dCTP. The K111 probe is 422 bp long and spans the CER:D22Z3 flanking sequence and the beginning of the LTR of K111. The K222 probe is 464 bp long and covers the pCER:D22Z8 flanking sequence and pro gene of K222. (B) DNA from the B-cell lines BJAB (having the 5′ end of K111) and IRA (lacking the 5′ end) as observed by PCR, were screened for K111 and K222 virus by slot blotting. DNA was cross-linked to PVDF membranes and screened for K111 and K222 using biotinylated probes. The probes were detected by chemiluminescence with HRP-conjugated streptavidin. The K111 probe, which targets the 5′ end of genomic K111, reacted with the DNA of BJAB cells but not IRA cells, confirming the lack of the 5′ end of the viral genome in IRA cells. The K222 probe reacted with the DNA of both BJAB and IRA cells, confirming that both cell lines have provirus K222, which is truncated at the 5′ end. Mouse DNA served as a negative control, and plasmids containing either K111 or K222 genomes were used as positive controls. The K111 probe did not react with the K222 plasmid and vice versa.
    Figure Legend Snippet: Detection of the K222 provirus in the genome of human cell lines by slot blot analysis. The DNA of human cell lines that were found to have or lack the 5′ end of K111 by PCR, and presumably contain the truncated K222 provirus, were screened for K111 and K222 by slot blot analyses. (A) Generation of K111 and K222-specific biotinylated probes. Probes were generated by PCR incorporation of biotin-labeled dCTP. The K111 probe is 422 bp long and spans the CER:D22Z3 flanking sequence and the beginning of the LTR of K111. The K222 probe is 464 bp long and covers the pCER:D22Z8 flanking sequence and pro gene of K222. (B) DNA from the B-cell lines BJAB (having the 5′ end of K111) and IRA (lacking the 5′ end) as observed by PCR, were screened for K111 and K222 virus by slot blotting. DNA was cross-linked to PVDF membranes and screened for K111 and K222 using biotinylated probes. The probes were detected by chemiluminescence with HRP-conjugated streptavidin. The K111 probe, which targets the 5′ end of genomic K111, reacted with the DNA of BJAB cells but not IRA cells, confirming the lack of the 5′ end of the viral genome in IRA cells. The K222 probe reacted with the DNA of both BJAB and IRA cells, confirming that both cell lines have provirus K222, which is truncated at the 5′ end. Mouse DNA served as a negative control, and plasmids containing either K111 or K222 genomes were used as positive controls. The K111 probe did not react with the K222 plasmid and vice versa.

    Techniques Used: Dot Blot, Polymerase Chain Reaction, Generated, Labeling, Sequencing, Negative Control, Plasmid Preparation

    10) Product Images from "The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA"

    Article Title: The functional interactome of PYHIN immune regulators reveals IFIX is a sensor of viral DNA

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20145808

    IFIX is a DNA-binding protein that functions as a viral DNA sensor A Biotinylated ISD was incubated with either recombinant GST-tagged IFIX-PY (aa 1–100) or IFIX-HIN200 (aa 200–492) and competed off with unlabeled ISD. DNA–protein complexes were resolved by non-denaturing PAGE, and biotinylated substrates were visualized. B Biotinylated ISD, pcDNA3.1, or Bam HI-linearized pcDNA3.1 were incubated with recombinant GST-tagged IFIX-HIN200 and competed off with unlabeled ISD. DNA–protein complexes were resolved as above. C Recombinant GST-tagged IFIX-HIN200 was incubated with a dsDNA array consisting of all possible 10mer oligonucleotide sequences, and DNA–protein binding was visualized with a fluorescent GST-specific antibody. To illustrate IFIX-HIN200 sequence preferences, fluorescence intensity is plotted against oligomer sequence content. D IFIX-GFP HEK293 cells were transfected with Cy3-labeled VACV 70mer and imaged by fluorescence confocal microscopy (315× zoom factor). White arrows indicate co-localization between IFIX-GFP and VACV 70mer. E Control or IFIX-GFP HEK293 cells were transfected with VACV 70mer, and total RNA was collected after 6 h. Relative induction of ifn-β was quantified by RT–qPCR. mRNA levels are normalized to cellular gapdh levels. The basal expression levels in the IFIX-inducible HEK293 cells for ifn-β (right bar at +Tet/-VACV70) and ifix (left bar at -Tet/-VAVC70) relative to gapdh were 2E-6 and 3E-6, respectively. This corresponds to raw C T values of ˜28 and 27 for ifn-β and ifix , respectively. Mean values ± SEM ( n = 3) are shown. **P ≤ 0.01 compared to transfected control cells and mock-transfected IFIX-GFP-expressing cells (Student's unpaired t -test; two-tailed). F GFP or GFP-IFIX HEK293 cells were infected with HSV-1 (MOI = 5) and cross-linked at 7 h post-infection. Cell lysates were subjected to α-GFP immunoaffinity isolation, and viral DNA binding was assessed by RT–PCR using HSV-1-specific genome sequences (top). Isolated GFP or GFP-IFIX (green arrows) was assessed by Western blotting (bottom). G Localization of IFIX-GFP in HSV-1-infected HEK293 cells (MOI = 1; 4 h post-infection) was visualized by fluorescence confocal microscopy. Viral protein ICP27: marker for infection. Scale bars, 5 μm.
    Figure Legend Snippet: IFIX is a DNA-binding protein that functions as a viral DNA sensor A Biotinylated ISD was incubated with either recombinant GST-tagged IFIX-PY (aa 1–100) or IFIX-HIN200 (aa 200–492) and competed off with unlabeled ISD. DNA–protein complexes were resolved by non-denaturing PAGE, and biotinylated substrates were visualized. B Biotinylated ISD, pcDNA3.1, or Bam HI-linearized pcDNA3.1 were incubated with recombinant GST-tagged IFIX-HIN200 and competed off with unlabeled ISD. DNA–protein complexes were resolved as above. C Recombinant GST-tagged IFIX-HIN200 was incubated with a dsDNA array consisting of all possible 10mer oligonucleotide sequences, and DNA–protein binding was visualized with a fluorescent GST-specific antibody. To illustrate IFIX-HIN200 sequence preferences, fluorescence intensity is plotted against oligomer sequence content. D IFIX-GFP HEK293 cells were transfected with Cy3-labeled VACV 70mer and imaged by fluorescence confocal microscopy (315× zoom factor). White arrows indicate co-localization between IFIX-GFP and VACV 70mer. E Control or IFIX-GFP HEK293 cells were transfected with VACV 70mer, and total RNA was collected after 6 h. Relative induction of ifn-β was quantified by RT–qPCR. mRNA levels are normalized to cellular gapdh levels. The basal expression levels in the IFIX-inducible HEK293 cells for ifn-β (right bar at +Tet/-VACV70) and ifix (left bar at -Tet/-VAVC70) relative to gapdh were 2E-6 and 3E-6, respectively. This corresponds to raw C T values of ˜28 and 27 for ifn-β and ifix , respectively. Mean values ± SEM ( n = 3) are shown. **P ≤ 0.01 compared to transfected control cells and mock-transfected IFIX-GFP-expressing cells (Student's unpaired t -test; two-tailed). F GFP or GFP-IFIX HEK293 cells were infected with HSV-1 (MOI = 5) and cross-linked at 7 h post-infection. Cell lysates were subjected to α-GFP immunoaffinity isolation, and viral DNA binding was assessed by RT–PCR using HSV-1-specific genome sequences (top). Isolated GFP or GFP-IFIX (green arrows) was assessed by Western blotting (bottom). G Localization of IFIX-GFP in HSV-1-infected HEK293 cells (MOI = 1; 4 h post-infection) was visualized by fluorescence confocal microscopy. Viral protein ICP27: marker for infection. Scale bars, 5 μm.

    Techniques Used: Binding Assay, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Protein Binding, Sequencing, Fluorescence, Transfection, Labeling, Confocal Microscopy, Quantitative RT-PCR, Expressing, Two Tailed Test, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Marker

    11) Product Images from "A General Method for Discovering Inhibitors of Protein-DNA Interactions Using Photonic Crystal Biosensors"

    Article Title: A General Method for Discovering Inhibitors of Protein-DNA Interactions Using Photonic Crystal Biosensors

    Journal: ACS chemical biology

    doi: 10.1021/cb800057j

    a) MazEF associates with its promoter sequence bound to the PC biosensor surface in a dose-dependent fashion. b) Preincubation of MazEF (1.8 μM) with its nonbiotinylated promoter sequence reduces the association of MazEF with the promoter-bound biosensor surface. c) Kinetics of MazEF (0.2 mg mL -1 ) binding to its own promoter sequence. A rapid increase in PWV shift is observed upon MazEF addition to the promoter-bound biosensor surface. In contrast, MazEF showed little affinity for a biotinylated alternating GC control DNA of the same length as its promoter sequence, similar to its association blocked sensor surface (no DNA). All error bars represent the calculated standard error ( n = 3).
    Figure Legend Snippet: a) MazEF associates with its promoter sequence bound to the PC biosensor surface in a dose-dependent fashion. b) Preincubation of MazEF (1.8 μM) with its nonbiotinylated promoter sequence reduces the association of MazEF with the promoter-bound biosensor surface. c) Kinetics of MazEF (0.2 mg mL -1 ) binding to its own promoter sequence. A rapid increase in PWV shift is observed upon MazEF addition to the promoter-bound biosensor surface. In contrast, MazEF showed little affinity for a biotinylated alternating GC control DNA of the same length as its promoter sequence, similar to its association blocked sensor surface (no DNA). All error bars represent the calculated standard error ( n = 3).

    Techniques Used: Sequencing, Binding Assay

    a) Schematic of the PC biosensor. A broadband LED illuminates the biosensor from the bottom, and reflected light is collected and transferred to a spectrometer where the PWV is measured. b) Image of PC biosensor films adhered to the bottom of black 384-well plates. c) Diagram of protein–DNA binding experiments performed with PC biosensors. Streptavidin-coated biosensors are used to bind biotinylated DNA oligomers, and a distinct peak wavelength of the reflected light is observed. After the addition of Starting Block (Pierce Biotechnologies), a DNA-binding protein is added, and a shift in the wavelength of reflected light is observed.
    Figure Legend Snippet: a) Schematic of the PC biosensor. A broadband LED illuminates the biosensor from the bottom, and reflected light is collected and transferred to a spectrometer where the PWV is measured. b) Image of PC biosensor films adhered to the bottom of black 384-well plates. c) Diagram of protein–DNA binding experiments performed with PC biosensors. Streptavidin-coated biosensors are used to bind biotinylated DNA oligomers, and a distinct peak wavelength of the reflected light is observed. After the addition of Starting Block (Pierce Biotechnologies), a DNA-binding protein is added, and a shift in the wavelength of reflected light is observed.

    Techniques Used: Binding Assay, Blocking Assay

    12) Product Images from "Affinity Purification of Binding miRNAs for Messenger RNA Fused with a Common Tag"

    Article Title: Affinity Purification of Binding miRNAs for Messenger RNA Fused with a Common Tag

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms150814753

    Over-expression of Argonaute 2 protein is to increasing the bingding force between miRNA and mRNA. ( A ) Biotinylated oligo DNA were not localization with P-body in cells; and ( B ) Flag/HA-Ago 2 were detected in the extracts of cells that transfected with Flag/HA-Ago 2 vector.
    Figure Legend Snippet: Over-expression of Argonaute 2 protein is to increasing the bingding force between miRNA and mRNA. ( A ) Biotinylated oligo DNA were not localization with P-body in cells; and ( B ) Flag/HA-Ago 2 were detected in the extracts of cells that transfected with Flag/HA-Ago 2 vector.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    The pull-down strategy of the determination of the microRNAs (miRNAs) that target to a specific gene. Extracts from the cells that over-expressed a tagged Argonaute protein are first incubated with biotinylated oligo DNA mix, and then affinity purified on streptavidin beads. MiRNAs are quantified by real time quantitative PCR (qRT-PCR).
    Figure Legend Snippet: The pull-down strategy of the determination of the microRNAs (miRNAs) that target to a specific gene. Extracts from the cells that over-expressed a tagged Argonaute protein are first incubated with biotinylated oligo DNA mix, and then affinity purified on streptavidin beads. MiRNAs are quantified by real time quantitative PCR (qRT-PCR).

    Techniques Used: Incubation, Affinity Purification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Antisense oligo DNAs of EGFP mRNA were labeled with biotin. ( A ) The secondary structures of the EGFP mRNA were predicted by software M-Fold; ( B ) The sequence of antisense oligo DNA targeting to EGFP mRNA were biotinylated at 3'-end; and ( C ) Biotinylated oligo DNA were detected by fluorescent microscope through Dylight 488-streptavidin.
    Figure Legend Snippet: Antisense oligo DNAs of EGFP mRNA were labeled with biotin. ( A ) The secondary structures of the EGFP mRNA were predicted by software M-Fold; ( B ) The sequence of antisense oligo DNA targeting to EGFP mRNA were biotinylated at 3'-end; and ( C ) Biotinylated oligo DNA were detected by fluorescent microscope through Dylight 488-streptavidin.

    Techniques Used: Labeling, Software, Sequencing, Microscopy

    13) Product Images from "Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes"

    Article Title: Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes

    Journal: eLife

    doi: 10.7554/eLife.17556

    Measurements of Sir3 binding to MonoN with BLI. ( A ) MonoN and DiN reconstitution using histone octamers containing histone H2A-K120C covalently linked to biotin-PEG2 maleimide. ( B ) Restriction enzyme protection assay of biotinylated DiN. ( C ) BLI assay of Sir3 binding to MonoN with differently biotinylated MonoN substrates yields identical results. The titration shows normalized equilibrium binding signal, integrating all modes of Sir3 binding to MonoN. H2A-biotin-PEG2 MonoN: Maleimide-PEG2-Biotin moiety covalently attached to histone H2A K120C SH group, 167bp-DNA-biotin: biotin moiety is covalently attached to the 5’-end at the beginning of DNA template (20 bp extension + 147 bp 601 positioning sequence). ( D ) Equilibrium binding signal to unmodified (dark blue) or acetylated (light blue) nucleosomes bearing H2A-biotin-PEG2 were measured by BLI at [Sir3] = 1.0–1.5 µM. Nucleosomes were loaded on streptavidin-coated BLI sensors to the same level before being used for Sir3 titration experiments. Histograms show the average and standard deviation of the equilibrium binding signal of the three BLI sensograms shown on the left. DOI: http://dx.doi.org/10.7554/eLife.17556.006
    Figure Legend Snippet: Measurements of Sir3 binding to MonoN with BLI. ( A ) MonoN and DiN reconstitution using histone octamers containing histone H2A-K120C covalently linked to biotin-PEG2 maleimide. ( B ) Restriction enzyme protection assay of biotinylated DiN. ( C ) BLI assay of Sir3 binding to MonoN with differently biotinylated MonoN substrates yields identical results. The titration shows normalized equilibrium binding signal, integrating all modes of Sir3 binding to MonoN. H2A-biotin-PEG2 MonoN: Maleimide-PEG2-Biotin moiety covalently attached to histone H2A K120C SH group, 167bp-DNA-biotin: biotin moiety is covalently attached to the 5’-end at the beginning of DNA template (20 bp extension + 147 bp 601 positioning sequence). ( D ) Equilibrium binding signal to unmodified (dark blue) or acetylated (light blue) nucleosomes bearing H2A-biotin-PEG2 were measured by BLI at [Sir3] = 1.0–1.5 µM. Nucleosomes were loaded on streptavidin-coated BLI sensors to the same level before being used for Sir3 titration experiments. Histograms show the average and standard deviation of the equilibrium binding signal of the three BLI sensograms shown on the left. DOI: http://dx.doi.org/10.7554/eLife.17556.006

    Techniques Used: Binding Assay, Titration, Sequencing, Standard Deviation

    14) Product Images from "Elevated levels of the second messenger c-di-GMP contribute to antimicrobial resistance of Pseudomonas aeruginosa"

    Article Title: Elevated levels of the second messenger c-di-GMP contribute to antimicrobial resistance of Pseudomonas aeruginosa

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12587

    Exogenous addition of c-di-GMP in vitro and in vivo elevated levels of c-di-GMP upon multicopy expression of PA4843 restore DNA-binding capability of BrlR obtained from total cells extracts of ΔsagS biofilm cells (A) Streptavidin magnetic bead DNA-binding assays using cell extracts obtained from Δ sagS and PAO1 biofilms harboring pMJT- brlR- V5/His 6 in the absence of presence of PA4843 expressed in trans . Binding assays were carried out using a total of 5pmol the BrlR-V5/His 6 protein and 1 pmol biotinylated P mexA or 1 pmol of biotinylated P mexE in wild-type and Δ sagS strains. Non-biotinylated P mexA and P mexE (P mexA -NB, P mexE -NB) were used as specific competitor DNA in 20-fold excess. BrlR binding to P mexA and P mexE was detected by immunoblot analysis using anti-V5 antibodies. +, indicates presence of specified components. Images on the right demonstrate BrlR to be present in similar amounts in aliquots removed prior to addition of streptavidin magnetic beads in Δ sagS and PAO1 biofilm samples (loading control). (B) Streptavidin magnetic bead DNA-binding assays using cell extracts obtained from Δ sagS and PAO1 harboring pMJT- brlR- V5/His 6 in the presence of increasing concentrations of c-di-GMP (0–200 pmol). Binding assays were carried out using a total of 5pmol the BrlR-V5/His 6 protein and 1 pmol biotinylated P mexA or 1 pmol of biotinylated P mexE in wild-type and Δ sagS strains. (C) BrlR-c-di-GMP binding assays. Pulldowns using biotinylated c-di-GMP immobilized to streptavidin magnetic beads demonstrate that BrlR produced by wild-type and Δ sagS biofilms is capable of c-di-GMP binding. Binding to biotinylated c-di-GMP was reduced by the addition of non-biotinylated c-di-GMP (c-di-GMP-NB). No binding was detected when BrlR produced by wild-type and Δ sagS biofilms overexpressing PA4843 was used. BrlR-c-di-GMP binding was detected by immunoblot analysis using anti-V5 antibodies. Experiments were repeated at least 4 times and representative images are shown.
    Figure Legend Snippet: Exogenous addition of c-di-GMP in vitro and in vivo elevated levels of c-di-GMP upon multicopy expression of PA4843 restore DNA-binding capability of BrlR obtained from total cells extracts of ΔsagS biofilm cells (A) Streptavidin magnetic bead DNA-binding assays using cell extracts obtained from Δ sagS and PAO1 biofilms harboring pMJT- brlR- V5/His 6 in the absence of presence of PA4843 expressed in trans . Binding assays were carried out using a total of 5pmol the BrlR-V5/His 6 protein and 1 pmol biotinylated P mexA or 1 pmol of biotinylated P mexE in wild-type and Δ sagS strains. Non-biotinylated P mexA and P mexE (P mexA -NB, P mexE -NB) were used as specific competitor DNA in 20-fold excess. BrlR binding to P mexA and P mexE was detected by immunoblot analysis using anti-V5 antibodies. +, indicates presence of specified components. Images on the right demonstrate BrlR to be present in similar amounts in aliquots removed prior to addition of streptavidin magnetic beads in Δ sagS and PAO1 biofilm samples (loading control). (B) Streptavidin magnetic bead DNA-binding assays using cell extracts obtained from Δ sagS and PAO1 harboring pMJT- brlR- V5/His 6 in the presence of increasing concentrations of c-di-GMP (0–200 pmol). Binding assays were carried out using a total of 5pmol the BrlR-V5/His 6 protein and 1 pmol biotinylated P mexA or 1 pmol of biotinylated P mexE in wild-type and Δ sagS strains. (C) BrlR-c-di-GMP binding assays. Pulldowns using biotinylated c-di-GMP immobilized to streptavidin magnetic beads demonstrate that BrlR produced by wild-type and Δ sagS biofilms is capable of c-di-GMP binding. Binding to biotinylated c-di-GMP was reduced by the addition of non-biotinylated c-di-GMP (c-di-GMP-NB). No binding was detected when BrlR produced by wild-type and Δ sagS biofilms overexpressing PA4843 was used. BrlR-c-di-GMP binding was detected by immunoblot analysis using anti-V5 antibodies. Experiments were repeated at least 4 times and representative images are shown.

    Techniques Used: In Vitro, In Vivo, Expressing, Binding Assay, Magnetic Beads, Produced

    15) Product Images from "BrlR from Pseudomonas aeruginosa is a c-di-GMP-responsive transcription factor"

    Article Title: BrlR from Pseudomonas aeruginosa is a c-di-GMP-responsive transcription factor

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12562

    BrlR is a c-di-GMP responsive DNA binding protein (A) BrlR-DNA gel mobility shift assays using (A) biotinylated P brlR DNA (0.5 pmol) in the absence and presence c-di-GMP. BrlR-DNA binding was detected by immunoblot analysis using anti-biotin antibodies. Arrowhead denotes shift. (B) Streptavidin BrlR-DNA binding assays using biotinylated P brlR DNA (0.5 pmol) in the absence and presence of increasing concentrations of c-di-GMP. A total of 25 pmol of purified His 6 V5-tagged BrlR was used. BrlR-DNA binding was detected by immunoblot analysis using anti-V5 antibodies. (C) BrlR binding to brlR promoter DNA can be outcompeted by increasing concentrations of non-biotinylated P brlR DNA (P brlR -NB). (D) BrlR binding to biotinylated P mexE (0.2 pmol) is enhanced by the presence of c-di-GMP as indicated using streptavidin binding assays. Experiments were carried out in triplicate and representative images are shown.
    Figure Legend Snippet: BrlR is a c-di-GMP responsive DNA binding protein (A) BrlR-DNA gel mobility shift assays using (A) biotinylated P brlR DNA (0.5 pmol) in the absence and presence c-di-GMP. BrlR-DNA binding was detected by immunoblot analysis using anti-biotin antibodies. Arrowhead denotes shift. (B) Streptavidin BrlR-DNA binding assays using biotinylated P brlR DNA (0.5 pmol) in the absence and presence of increasing concentrations of c-di-GMP. A total of 25 pmol of purified His 6 V5-tagged BrlR was used. BrlR-DNA binding was detected by immunoblot analysis using anti-V5 antibodies. (C) BrlR binding to brlR promoter DNA can be outcompeted by increasing concentrations of non-biotinylated P brlR DNA (P brlR -NB). (D) BrlR binding to biotinylated P mexE (0.2 pmol) is enhanced by the presence of c-di-GMP as indicated using streptavidin binding assays. Experiments were carried out in triplicate and representative images are shown.

    Techniques Used: Binding Assay, Mobility Shift, Purification

    BrlR binds to a palindromic DNA spacer sequence )). S, Synechocystis sp.; Ec, Escherichia coli ; BS, Bacillus subtilis ; Pa, Pseudomonas aeruginosa ; Sl, Streptomyces lividans )). (C) DNA gel mobility shift assays indicating binding of BrlR to the putative BrlR-DNA binding sequence. prom, DNA sequence spanning the -10 and -35 promoter elements (underlined); prom-10, DNA sequence lacking the -10 region; prom-35, DNA sequence lacking the -35 region; prom-19 bp, 19 bp spacer region located between the -10 and -35 promoter elements; prom-mut1, 19 bp spacer region located between the -10 and -35 promoter elements in which the G’s on the right side of the palindrome have been substituted with T’s; prom-mut2, 19bp-long sequence in part of a portion of the 19 bp spacer region and a DNA sequence located 119-128 bp upstream of the ATG start codon. A total of 5 pmol purified His 6 V5-tagged BrlR and 2.5 pmol per each biotinylated DNA sequence was used. BrlR-DNA binding was detected by immunoblot analysis using anti-biotin antibodies. Black arrowheads indicate specific shift. -, no DNA sequences or protein added. Total cell extract obtained from ΔbrlR was used as control. (D) MEME-derived BrlR-DNA binding motif common to BrlR-DNA binding sequence obtained in (A-B) and promoter sequences of the mexAB-oprM and mexEF-oprN operons.
    Figure Legend Snippet: BrlR binds to a palindromic DNA spacer sequence )). S, Synechocystis sp.; Ec, Escherichia coli ; BS, Bacillus subtilis ; Pa, Pseudomonas aeruginosa ; Sl, Streptomyces lividans )). (C) DNA gel mobility shift assays indicating binding of BrlR to the putative BrlR-DNA binding sequence. prom, DNA sequence spanning the -10 and -35 promoter elements (underlined); prom-10, DNA sequence lacking the -10 region; prom-35, DNA sequence lacking the -35 region; prom-19 bp, 19 bp spacer region located between the -10 and -35 promoter elements; prom-mut1, 19 bp spacer region located between the -10 and -35 promoter elements in which the G’s on the right side of the palindrome have been substituted with T’s; prom-mut2, 19bp-long sequence in part of a portion of the 19 bp spacer region and a DNA sequence located 119-128 bp upstream of the ATG start codon. A total of 5 pmol purified His 6 V5-tagged BrlR and 2.5 pmol per each biotinylated DNA sequence was used. BrlR-DNA binding was detected by immunoblot analysis using anti-biotin antibodies. Black arrowheads indicate specific shift. -, no DNA sequences or protein added. Total cell extract obtained from ΔbrlR was used as control. (D) MEME-derived BrlR-DNA binding motif common to BrlR-DNA binding sequence obtained in (A-B) and promoter sequences of the mexAB-oprM and mexEF-oprN operons.

    Techniques Used: Sequencing, Mobility Shift, Binding Assay, Purification, Derivative Assay

    16) Product Images from "Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein ▿ Intracellular Development by a Previously Unidentified Protein ▿ †"

    Article Title: Regulation of Type IV Secretion Apparatus Genes during Ehrlichia chaffeensis Intracellular Development by a Previously Unidentified Protein ▿ Intracellular Development by a Previously Unidentified Protein ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01813-07

    Identification of an E. chaffeensis protein bound to the DNA probes derived from sequences upstream of virBD loci. (A) EMSA of native E. chaffeensis proteins bound to the biotinylated DNA probes derived from sequences upstream of sodB and virB9-2 . Native
    Figure Legend Snippet: Identification of an E. chaffeensis protein bound to the DNA probes derived from sequences upstream of virBD loci. (A) EMSA of native E. chaffeensis proteins bound to the biotinylated DNA probes derived from sequences upstream of sodB and virB9-2 . Native

    Techniques Used: Derivative Assay

    17) Product Images from "Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators"

    Article Title: Cohesins localize with CTCF at the KSHV latency control region and at cellular c-myc and H19/Igf2 insulators

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2008.1

    Mapping CTCF-binding sites upstream of ORF73. ( A ) DNA affinity purification assay with biotinylated DNA from KSHV 126 841–127 840 or a control 1000-bp fragment from pBKS. BCBL1 nuclear extract was incubated with biotinylated DNA and bound proteins
    Figure Legend Snippet: Mapping CTCF-binding sites upstream of ORF73. ( A ) DNA affinity purification assay with biotinylated DNA from KSHV 126 841–127 840 or a control 1000-bp fragment from pBKS. BCBL1 nuclear extract was incubated with biotinylated DNA and bound proteins

    Techniques Used: Binding Assay, Affinity Purification, Incubation

    18) Product Images from "Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries"

    Article Title: Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0400678101

    Cloning of active chromatin sequences. We developed a strategy to create genomic DNA libraries containing sequences flanking DNaseI cut sites introduced into nuclear chromatin under limiting (hypersensitive) conditions. After DNA purification, free DNA ends are enzymatically repaired and ligated to a biotinylated linker adaptor. The DNA sample is then fragmented further with a four-cutter enzyme ( Nla III). At this stage, the genome has been partitioned into two predominant fragment populations: Nla III– Nla III fragments (derived from the non-DNaseI cut background) and Nla III-adaptor fragments (carrying for DNaseI cut sites). Adapted DNA is efficiently isolated on paramagnetic streptavidin-coated beads, whereas Nla III– Nla III background fragments are cleansed. A second linker adaptor is then appended to the Nla III end of captured DNA, and the product is released from the beads. This DNaseI cut site-enriched population is enriched and is retained for the subsequent subtraction step. A DNaseI cut site-depleted population is prepared by further fragmenting DNaseI-treated genomic DNA with a four-cutter that leaves a 3′ overhang (e.g., Nla III). Further digestion of this sample with Exonuclease III followed by mung bean nuclease will preserve the Nla III– Nla III fragments (which are resistant to processive degradation), whereas fragments with DNaseI cut ends will be efficiently eliminated. The residual remaining population of DNaseI cut site-depleted DNA is then heavily biotinylated. An excess of this population is mixed with the DNaseI cut site-enriched population, and the sample is denatured and is slowly reannealed. Nonbiotinylated fragments generated by repeated DNaseI cleavage events at or around the same genomic coordinate (i.e., a hypersensitive site) will be more likely to self-anneal than find a partner in the DNaseI cut site-depleted population. Sites that have only been cut once (i.e., due to non-HS-specific cutting or to genomic shear) will form heteroduplexes. Extraction of the mixture with paramagnetic beads isolates the nonbiotinylated homoduplexes that are now further enriched in DNaseI hypersensitive sites. This population is PCR-amplified and cloned to make the genomic ACS libraries.
    Figure Legend Snippet: Cloning of active chromatin sequences. We developed a strategy to create genomic DNA libraries containing sequences flanking DNaseI cut sites introduced into nuclear chromatin under limiting (hypersensitive) conditions. After DNA purification, free DNA ends are enzymatically repaired and ligated to a biotinylated linker adaptor. The DNA sample is then fragmented further with a four-cutter enzyme ( Nla III). At this stage, the genome has been partitioned into two predominant fragment populations: Nla III– Nla III fragments (derived from the non-DNaseI cut background) and Nla III-adaptor fragments (carrying for DNaseI cut sites). Adapted DNA is efficiently isolated on paramagnetic streptavidin-coated beads, whereas Nla III– Nla III background fragments are cleansed. A second linker adaptor is then appended to the Nla III end of captured DNA, and the product is released from the beads. This DNaseI cut site-enriched population is enriched and is retained for the subsequent subtraction step. A DNaseI cut site-depleted population is prepared by further fragmenting DNaseI-treated genomic DNA with a four-cutter that leaves a 3′ overhang (e.g., Nla III). Further digestion of this sample with Exonuclease III followed by mung bean nuclease will preserve the Nla III– Nla III fragments (which are resistant to processive degradation), whereas fragments with DNaseI cut ends will be efficiently eliminated. The residual remaining population of DNaseI cut site-depleted DNA is then heavily biotinylated. An excess of this population is mixed with the DNaseI cut site-enriched population, and the sample is denatured and is slowly reannealed. Nonbiotinylated fragments generated by repeated DNaseI cleavage events at or around the same genomic coordinate (i.e., a hypersensitive site) will be more likely to self-anneal than find a partner in the DNaseI cut site-depleted population. Sites that have only been cut once (i.e., due to non-HS-specific cutting or to genomic shear) will form heteroduplexes. Extraction of the mixture with paramagnetic beads isolates the nonbiotinylated homoduplexes that are now further enriched in DNaseI hypersensitive sites. This population is PCR-amplified and cloned to make the genomic ACS libraries.

    Techniques Used: Clone Assay, DNA Purification, Derivative Assay, Isolation, Generated, Polymerase Chain Reaction, Amplification

    19) Product Images from "Regulation of Bacterial DNA Packaging in Early Stationary Phase by Competitive DNA Binding of Dps and IHF"

    Article Title: Regulation of Bacterial DNA Packaging in Early Stationary Phase by Competitive DNA Binding of Dps and IHF

    Journal: Scientific Reports

    doi: 10.1038/srep18146

    Transverse magnetic tweezers setup and Dps mediated DNA condensation. ( a ) Figure shows an illustration of single-DNA stretching experiment using magnetic tweezers. One biotinylated DNA end is attached to a streptavidin functionalised coverslip and the other biotinylated DNA end attached to a streptavidin coated paramagnetic bead. In the shaded area of 2 micrometers from the coverslip edge, bead image cannot be obtained. Force is exerted by using a permanent magnet and force is adjusted by moving the position of magnet. At different forces, the corresponding extension of DNA is recorded. ( b ) Force-extension curves obtained on a 48,502 bp λ-DNA during force-decrease (solid symbols) and subsequent force-increase scan (open symbols) before and after incubated with 50 nM Dps, 500 nM Dps and 5000 nM Dps in 50 mM KCl, pH 7.5 at 23 °C. Black data shows force extension curve of naked DNA as control where force-decreased and force increased curves overlapped. The black solid curve is a fitting curve by the Worm-like chain DNA polymer model with a persistence length of 50 nm using the Marko-Siggia formula 35 36 . Force extension curves after protein incubation are plotted with coloured symbols, coloured lines are connecting lines between each data points for better presentation. Data obtained from three independent experiments at each concentration are shown in the same colour indicated by solid, dashed, and dotted connecting lines. The non-overlapping force-decreased and force increased curves shows that force-extension curves of the DNA interacting with Dps do not reach equilibrium at our force-scanning experimental time scale.
    Figure Legend Snippet: Transverse magnetic tweezers setup and Dps mediated DNA condensation. ( a ) Figure shows an illustration of single-DNA stretching experiment using magnetic tweezers. One biotinylated DNA end is attached to a streptavidin functionalised coverslip and the other biotinylated DNA end attached to a streptavidin coated paramagnetic bead. In the shaded area of 2 micrometers from the coverslip edge, bead image cannot be obtained. Force is exerted by using a permanent magnet and force is adjusted by moving the position of magnet. At different forces, the corresponding extension of DNA is recorded. ( b ) Force-extension curves obtained on a 48,502 bp λ-DNA during force-decrease (solid symbols) and subsequent force-increase scan (open symbols) before and after incubated with 50 nM Dps, 500 nM Dps and 5000 nM Dps in 50 mM KCl, pH 7.5 at 23 °C. Black data shows force extension curve of naked DNA as control where force-decreased and force increased curves overlapped. The black solid curve is a fitting curve by the Worm-like chain DNA polymer model with a persistence length of 50 nm using the Marko-Siggia formula 35 36 . Force extension curves after protein incubation are plotted with coloured symbols, coloured lines are connecting lines between each data points for better presentation. Data obtained from three independent experiments at each concentration are shown in the same colour indicated by solid, dashed, and dotted connecting lines. The non-overlapping force-decreased and force increased curves shows that force-extension curves of the DNA interacting with Dps do not reach equilibrium at our force-scanning experimental time scale.

    Techniques Used: Incubation, Concentration Assay

    20) Product Images from "A Structure-Based Mechanism for DNA Entry into the Cohesin Ring"

    Article Title: A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

    Journal: bioRxiv

    doi: 10.1101/2020.04.21.052944

    Cohesin ATPase Head Engagement Leads to a DNA ‘Gripping’ State (A) Schematic, purification and labeling of untagged, and fluorescent tagged wild type (WT) and Walker B mutant (EQ) cohesin to measure FRET between the Psm1 and Psm3 ATPase heads. Purified and labeled complexes were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue (CBB) staining or in gel fluorescence detection. (B) Head FRET efficiencies of EQ-cohesin with the indicated additions was calculated by dividing the Alexa 647 intensity at its emission peak with the sum of Alexa 647 and Dy547 intensities. Results from three independent repeats of the experiment, their means and standard deviations are shown. (C) Head FRET efficiencies of wild type cohesin in the presence of the Mis4-Ssl3 loader, a 3 kb circular plasmid DNA and the indicated nucleotides and phosphate analogs was measured as in (B). Results from four independent repeats of the experiment, their means and standard deviations are shown. (D) Schematic of the DNA gripping experiment in which biotinylated linear dsDNA is immobilized on streptavidin-coated magnetic beads. Cohesin, loader and the indicated nucleotides were added. After the incubation, beads were washed with buffer containing 135 mM NaCl. Bound protein was analyzed by SDS-PAGE and immunoblotting while the DNA was visualized by agarose gel electrophoresis. (E) Salt sensitivity of cohesin-DNA complexes following assembly with hydrolyzable or non-hydrolyzable ATP, on linear DNA and on DNA loops. Following the incubation, beads were washed with buffer containing 135 mM or 500 mM NaCl and products analyzed as in (D).
    Figure Legend Snippet: Cohesin ATPase Head Engagement Leads to a DNA ‘Gripping’ State (A) Schematic, purification and labeling of untagged, and fluorescent tagged wild type (WT) and Walker B mutant (EQ) cohesin to measure FRET between the Psm1 and Psm3 ATPase heads. Purified and labeled complexes were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue (CBB) staining or in gel fluorescence detection. (B) Head FRET efficiencies of EQ-cohesin with the indicated additions was calculated by dividing the Alexa 647 intensity at its emission peak with the sum of Alexa 647 and Dy547 intensities. Results from three independent repeats of the experiment, their means and standard deviations are shown. (C) Head FRET efficiencies of wild type cohesin in the presence of the Mis4-Ssl3 loader, a 3 kb circular plasmid DNA and the indicated nucleotides and phosphate analogs was measured as in (B). Results from four independent repeats of the experiment, their means and standard deviations are shown. (D) Schematic of the DNA gripping experiment in which biotinylated linear dsDNA is immobilized on streptavidin-coated magnetic beads. Cohesin, loader and the indicated nucleotides were added. After the incubation, beads were washed with buffer containing 135 mM NaCl. Bound protein was analyzed by SDS-PAGE and immunoblotting while the DNA was visualized by agarose gel electrophoresis. (E) Salt sensitivity of cohesin-DNA complexes following assembly with hydrolyzable or non-hydrolyzable ATP, on linear DNA and on DNA loops. Following the incubation, beads were washed with buffer containing 135 mM or 500 mM NaCl and products analyzed as in (D).

    Techniques Used: Purification, Labeling, Mutagenesis, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Fluorescence, Plasmid Preparation, Magnetic Beads, Incubation, Agarose Gel Electrophoresis

    21) Product Images from "Efficient preparation of internally modified single-molecule constructs using nicking enzymes"

    Article Title: Efficient preparation of internally modified single-molecule constructs using nicking enzymes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1004

    Schematic representation of the internal labeling method. ( a ) A DNA sequence which incorporates five equally spaced BbvCI recognition sites (black triangles) is nicked only at one of the two strands using either the nicking enzyme Nt.BbvCI or Nb.BbvCI. This results in the formation of short 15–16 bases long fragments. Denaturation and subsequent hybridization, in the presence of a DNA strand (shown in red) that is complementary to the resulting 63 bp gap and that carries the desired internal modifications (e.g. two or six biotins as depicted), lead to an efficient replacement of the original fragments with the labeled fragment. The spacing between the internal modifications of 10–11 bp ensures that they extrude in the same direction from the DNA. ( b ) Model of a streptavidin tetramer bound to an internally biotinylated DNA molecule (Streptavidin PDB id: 1MK5; the bound monomer is illustrated as a yellow ribbon while for the other subunits the surface representation was used. DNA PDB id: 2BNA). The attachment of the streptavidin tetramer to only one of the biotins was arbitrarily chosen.
    Figure Legend Snippet: Schematic representation of the internal labeling method. ( a ) A DNA sequence which incorporates five equally spaced BbvCI recognition sites (black triangles) is nicked only at one of the two strands using either the nicking enzyme Nt.BbvCI or Nb.BbvCI. This results in the formation of short 15–16 bases long fragments. Denaturation and subsequent hybridization, in the presence of a DNA strand (shown in red) that is complementary to the resulting 63 bp gap and that carries the desired internal modifications (e.g. two or six biotins as depicted), lead to an efficient replacement of the original fragments with the labeled fragment. The spacing between the internal modifications of 10–11 bp ensures that they extrude in the same direction from the DNA. ( b ) Model of a streptavidin tetramer bound to an internally biotinylated DNA molecule (Streptavidin PDB id: 1MK5; the bound monomer is illustrated as a yellow ribbon while for the other subunits the surface representation was used. DNA PDB id: 2BNA). The attachment of the streptavidin tetramer to only one of the biotins was arbitrarily chosen.

    Techniques Used: Labeling, Sequencing, Hybridization

    Site-specific attachment of Q-dots to internally biotinylated DNA. ( a ) Band-shift assay of Q-dot binding to DNA. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb. (Lane 1) pNLrep after digestion with BamHI, PspOMI and Nt.BbvCI and internal biotinylation with oligomer biotinx2 ( Figure 2 a). (Lane 2) Sample from Lane 1 with 5-fold molar excess of streptavidin coated Q-dots added. (Lane 3) Sample containing Q-dots only. Symbols on the right side indicate Q-dots (yellow spheres), the short biotinylated fragment (red line) and the long non-biotinylated fragment (blue line). ( b and c ) AFM images of Q-dots bound to DNA. The colour scale corresponds to a height-range of 1 nm, and the scale bar corresponds to 100 nm. ( d ) Histogram of the Q-dot position measured from the nearest DNA end. The expected Q-dot position at 920 bp (310 nm) (blue dashed line) is within the double confidence interval (light grey band) of the experimentally determined mean (290 ± 20 nm, red dashed line).
    Figure Legend Snippet: Site-specific attachment of Q-dots to internally biotinylated DNA. ( a ) Band-shift assay of Q-dot binding to DNA. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb. (Lane 1) pNLrep after digestion with BamHI, PspOMI and Nt.BbvCI and internal biotinylation with oligomer biotinx2 ( Figure 2 a). (Lane 2) Sample from Lane 1 with 5-fold molar excess of streptavidin coated Q-dots added. (Lane 3) Sample containing Q-dots only. Symbols on the right side indicate Q-dots (yellow spheres), the short biotinylated fragment (red line) and the long non-biotinylated fragment (blue line). ( b and c ) AFM images of Q-dots bound to DNA. The colour scale corresponds to a height-range of 1 nm, and the scale bar corresponds to 100 nm. ( d ) Histogram of the Q-dot position measured from the nearest DNA end. The expected Q-dot position at 920 bp (310 nm) (blue dashed line) is within the double confidence interval (light grey band) of the experimentally determined mean (290 ± 20 nm, red dashed line).

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay

    Internal modification and religation efficiencies. ( a ) Polyacrylamide gel electrophoresis of DNA samples in the course of the internal labeling procedure. (Lane 1) pNLrep after digestion with MluI and AatII yielding a 0.8-kb fragment (red line) that carries the region to be replaced as well as a 0.4-kb (light blue line) and a 5-kbp fragment (dark blue line). (Lane 2) pNLrep after simultaneous digestion with MluI, AatII and Nt.BbvCI. The nicking of the 0.8-kb fragment (represented by the fragmented red line) can be seen as a slight mobility decrease. (Lane 3) Sample from lane 2 after column purification, which leads to gap formation within the 0.8-kb fragment causing a large mobility alteration (gapped red line). (Lane 4) Sample from lane 2 after the replacement reaction with oligo biotinx2, during which the 0.8-kb fragment becomes internally biotinylated, and subsequent column purification. The inserted oligo is stably bound and therefore displays the same mobility as the nicked fragment in lane 2. (Lane 5) Sample from lane 4 with > 10-fold molar excess of streptavidin added. (Lane 6) Pulldown assay with sample from lane 4 (see ‘Materials and Methods' section). (Lanes 0, 7) 100 bp step DNA ladder, starting at 400 bp with an additional 517 bp band. ( b ) (Lane 1) pNLrep. (Lane 2) pNLrep after nicking and internal biotinylation with oligo biotinx2. (Lane 3) Sample from lane 2 after ligation. (Lane 4) pNLrep after internal biotinylation with 5′-phosphorylated biotinx2 oligo and religation. (Lane 5) pNLrep after nicking with Nt.BbvCI and religation. Positions of supercoiled, nicked and linearized plasmid species are indicated by corresponding symbols at the right side. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb.
    Figure Legend Snippet: Internal modification and religation efficiencies. ( a ) Polyacrylamide gel electrophoresis of DNA samples in the course of the internal labeling procedure. (Lane 1) pNLrep after digestion with MluI and AatII yielding a 0.8-kb fragment (red line) that carries the region to be replaced as well as a 0.4-kb (light blue line) and a 5-kbp fragment (dark blue line). (Lane 2) pNLrep after simultaneous digestion with MluI, AatII and Nt.BbvCI. The nicking of the 0.8-kb fragment (represented by the fragmented red line) can be seen as a slight mobility decrease. (Lane 3) Sample from lane 2 after column purification, which leads to gap formation within the 0.8-kb fragment causing a large mobility alteration (gapped red line). (Lane 4) Sample from lane 2 after the replacement reaction with oligo biotinx2, during which the 0.8-kb fragment becomes internally biotinylated, and subsequent column purification. The inserted oligo is stably bound and therefore displays the same mobility as the nicked fragment in lane 2. (Lane 5) Sample from lane 4 with > 10-fold molar excess of streptavidin added. (Lane 6) Pulldown assay with sample from lane 4 (see ‘Materials and Methods' section). (Lanes 0, 7) 100 bp step DNA ladder, starting at 400 bp with an additional 517 bp band. ( b ) (Lane 1) pNLrep. (Lane 2) pNLrep after nicking and internal biotinylation with oligo biotinx2. (Lane 3) Sample from lane 2 after ligation. (Lane 4) pNLrep after internal biotinylation with 5′-phosphorylated biotinx2 oligo and religation. (Lane 5) pNLrep after nicking with Nt.BbvCI and religation. Positions of supercoiled, nicked and linearized plasmid species are indicated by corresponding symbols at the right side. (Lane 0) 1 kb step DNA ladder with the shortest fragment starting at 1 kb.

    Techniques Used: Modification, Polyacrylamide Gel Electrophoresis, Labeling, Purification, Stable Transfection, Ligation, Plasmid Preparation

    ssDNA to dsDNA ligation at nicking-enzyme-generated overhangs. ( a ) Schematic representation of overhang generation. A BbvCI recognition site (blue letters) was incorporated near the DNA end in such a way that nicking with Nt.BbvCI generates a 10-bp fragment at the 5′-end. ( b ) Agarose gel of DNA fragments, ligation products and streptavidin-induced band shifts. The biotinylated 40-bp hairpin, the 430-bp dsDNA handle and streptavidin are represented by a blue, red and green symbol, respectively. `Lig.' indicates where a ligation for 1 h at room temperature was carried out. Positions of the reaction products are marked at the right side. (Lanes 1–5) reaction products for the 4 nt overhang generated by BstXI. (Lanes 6–10) reaction products for the 10 nt overhang generated by Nt.BbvCI. The lane in the middle is a 100-bp size marker ladder with the shortest fragment starting at 100-bp and 100-bp size difference between all subsequent fragments (and an additional band at 517 bp). The success of the ssDNA to dsDNA ligation was confirmed by the streptavidin-induced band-shift, in which the desired product specifically shifted only in the case where a 10 nt 3′-overhang had been used. ( c ) Magnetic tweezers experiment with the generated hairpin construct. The molecule was held at the critical force where the closed and the opened states of the hairpin (as illustrated by the sketches) were nearly equally populated. The change in height between the two states was ≈38 nm as expected for a 40-nt hairpin.
    Figure Legend Snippet: ssDNA to dsDNA ligation at nicking-enzyme-generated overhangs. ( a ) Schematic representation of overhang generation. A BbvCI recognition site (blue letters) was incorporated near the DNA end in such a way that nicking with Nt.BbvCI generates a 10-bp fragment at the 5′-end. ( b ) Agarose gel of DNA fragments, ligation products and streptavidin-induced band shifts. The biotinylated 40-bp hairpin, the 430-bp dsDNA handle and streptavidin are represented by a blue, red and green symbol, respectively. `Lig.' indicates where a ligation for 1 h at room temperature was carried out. Positions of the reaction products are marked at the right side. (Lanes 1–5) reaction products for the 4 nt overhang generated by BstXI. (Lanes 6–10) reaction products for the 10 nt overhang generated by Nt.BbvCI. The lane in the middle is a 100-bp size marker ladder with the shortest fragment starting at 100-bp and 100-bp size difference between all subsequent fragments (and an additional band at 517 bp). The success of the ssDNA to dsDNA ligation was confirmed by the streptavidin-induced band-shift, in which the desired product specifically shifted only in the case where a 10 nt 3′-overhang had been used. ( c ) Magnetic tweezers experiment with the generated hairpin construct. The molecule was held at the critical force where the closed and the opened states of the hairpin (as illustrated by the sketches) were nearly equally populated. The change in height between the two states was ≈38 nm as expected for a 40-nt hairpin.

    Techniques Used: Ligation, Generated, Agarose Gel Electrophoresis, Marker, Electrophoretic Mobility Shift Assay, Construct

    22) Product Images from "IFI16 filament formation in salivary epithelial cells shapes the anti-IFI16 immune response in Sjögren’s syndrome"

    Article Title: IFI16 filament formation in salivary epithelial cells shapes the anti-IFI16 immune response in Sjögren’s syndrome

    Journal: JCI Insight

    doi: 10.1172/jci.insight.120179

    IFI16•dsDNA filaments are released from epithelial cells following exposure to cytotoxic lymphocyte granule contents. ( A and B ) IFN-treated human salivary gland (HSG) cells were transfected with biotinylated DNA to induce IFI16 filament formation and were then mock treated ( A ) or exposed to YT cell granule contents (GC) for 3 hours ( B ). Cells were fixed and stained with DAPI (blue), anti-IFI16 antibody (green), and Streptavidin-DyLight 594 (red). ( C ) Induction of apoptosis by GC treatment was confirmed by Western blotting for caspase 3, demonstrating intact (closed arrow) and cleaved (open arrow) forms. ( D ) Following GC treatment, samples of cell lysate (top panel) and supernatant (middle panel) were collected and analyzed in parallel for IFI16 and DNA content by blotting with anti-IFI16 antibody and StrepTactin-HRP. Biotinylated DNA was isolated from the supernatant of treated cells using Streptavidin DynaBeads, and the presence of IFI16•dsDNA interaction in the supernatant was confirmed by blotting for each ( D , bottom panel). Scale bars: 10 μM. Data are representative of results of 3 separate experiments.
    Figure Legend Snippet: IFI16•dsDNA filaments are released from epithelial cells following exposure to cytotoxic lymphocyte granule contents. ( A and B ) IFN-treated human salivary gland (HSG) cells were transfected with biotinylated DNA to induce IFI16 filament formation and were then mock treated ( A ) or exposed to YT cell granule contents (GC) for 3 hours ( B ). Cells were fixed and stained with DAPI (blue), anti-IFI16 antibody (green), and Streptavidin-DyLight 594 (red). ( C ) Induction of apoptosis by GC treatment was confirmed by Western blotting for caspase 3, demonstrating intact (closed arrow) and cleaved (open arrow) forms. ( D ) Following GC treatment, samples of cell lysate (top panel) and supernatant (middle panel) were collected and analyzed in parallel for IFI16 and DNA content by blotting with anti-IFI16 antibody and StrepTactin-HRP. Biotinylated DNA was isolated from the supernatant of treated cells using Streptavidin DynaBeads, and the presence of IFI16•dsDNA interaction in the supernatant was confirmed by blotting for each ( D , bottom panel). Scale bars: 10 μM. Data are representative of results of 3 separate experiments.

    Techniques Used: Transfection, Staining, Western Blot, Isolation

    23) Product Images from "A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention"

    Article Title: A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention

    Journal: Journal of Virology

    doi: 10.1128/JVI.02143-18

    PcG proteins bind to replicating HCMV genomes. (A) HFF cells were infected with AD169 at an MOI of 1.5. At 72 hpi, newly synthesized DNA was labeled with EdU (10 μM) for 4 h, and Alexa 488 azide was conjugated by click chemistry. The viral protein pUL44 was visualized by antibody staining. The samples were analyzed by confocal microscopy. (B) Graph representing the percentage of cells positive for EdU only or for both EdU and pUL44 (mock, n  = 150; infected, n  = 150). (C) HFF cells were infected with AD169 at an MOI of 1.5 and used for accelerated native isolation of proteins on nascent DNA (aniPOND). At 72 hpi, cells were labeled for 4 h and biotinylated by click chemistry for affinity purification. No click reaction served as the negative control.
    Figure Legend Snippet: PcG proteins bind to replicating HCMV genomes. (A) HFF cells were infected with AD169 at an MOI of 1.5. At 72 hpi, newly synthesized DNA was labeled with EdU (10 μM) for 4 h, and Alexa 488 azide was conjugated by click chemistry. The viral protein pUL44 was visualized by antibody staining. The samples were analyzed by confocal microscopy. (B) Graph representing the percentage of cells positive for EdU only or for both EdU and pUL44 (mock, n  = 150; infected, n  = 150). (C) HFF cells were infected with AD169 at an MOI of 1.5 and used for accelerated native isolation of proteins on nascent DNA (aniPOND). At 72 hpi, cells were labeled for 4 h and biotinylated by click chemistry for affinity purification. No click reaction served as the negative control.

    Techniques Used: Infection, Synthesized, Labeling, Staining, Confocal Microscopy, Isolation, Affinity Purification, Negative Control

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    Incubation:

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    Binding Assay:

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    SDS Page:

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    Blocking Assay:

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  • 99
    Thermo Fisher biotinylated bsa
    Visualizing unwinding of individual DNA molecules using fluorescent SSB protein and TIRF microscopy. (A) Top : Diagram of phage λ DNA molecule with the indicated number of biotin groups incorporated in each 12-nt cos overhang. Middle : Illustration of <t>biotinylated</t> λ DNA attached at both ends via <t>biotin–streptavidin</t> linkage. Bottom : Image of an actual λ DNA molecule attached to the glass surface, stained with YO-PRO-1 (100 n M ), and illuminated with a 488 nm laser. The image is false colored in green and the attachment points to the glass surface are indicated. (B) The process required to construct a flow cell containing three separate single-channels. The steps highlighted are equivalent to those described in Section 4.1. (C) The flow cell from (B) mounted onto the objective; biotinylated lambda DNA was injected under buffer flow, permitting attachment of both ends to the surface. Unwinding tracks are visualized by binding of AF488-SSB G26C ( green ) to ssDNA regions. (D) Schematic representation of a TIRF microscope capable of visualizing DNA unwinding by RecQ by monitoring signal from both DNA and fluorescent SSB simultaneously. As shown in (C) the flow cell is mounted onto a 100× oil-immersion objective. The fluorescent SSB and DNA are excited by two lasers; 488 and 561 nm, respectively, and emission measured, via dichroic mirrors (M1 and M2). The deconvoluted emission is then directed onto different areas of a CCD camera generating a signal corresponding to either SSB or DNA. The lasers are operated using a custom LABview VI program to coordinate excitation with image acquisition so that the sample is illuminated only during the exposure times. Panels (A) and (D): From . Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding. Proceedings of the National Academy of Sciences of the United States of America, 112 (50), E6852 – E6861 .
    Biotinylated Bsa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher biotinylated t1 4
    Three viral noncoding RNAs were identified and confirmed to be directly associated with K8 protein. (A) CLIP-seq reads mapped to the KSHV genome. Enlargement of the K8 cross-linked RNA in the KSHV genomic location showed two peaks <t>(T1.4</t> RNA and PAN RNA) from the forward strain (left) and one peak (T0.7 RNA) from the reverse strain (right). (B and C) PAN (B) and T1.4 (C) RNAs and their truncation mutants were synthesized in vitro and labeled with biotin. An in vitro RNA pulldown assay was performed to evaluate the abilities of the RNAs and their mutants to be bound by K8. <t>Biotinylated</t> transcripts were mixed with purified GST-K8 protein, and the RNA-protein complexes were precipitated with streptavidin-coupled Dynabeads. The precipitates were analyzed by Western blotting for RNA-bound GST-K8 using anti-GST antibody and by Northern blotting for RNAs using the chemiluminescent nucleic acid detection module.
    Biotinylated T1 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher 㠎 â 1 4 m biotinylated dna
    Cortisol has a modest impact on <t>RBPJ/DNA</t> interactions. Primary osteoblast-enriched cells were seeded on BSA or DLL1, confluent cultures maintained for 24 h in the absence of serum and then exposed to vehicle or cortisol 1 <t>μM</t> for 6 h. Nuclear proteins were extracted and binding reactions carried out with a <t>biotinylated</t> oligonucleotide containing an RBPJ consensus sequence from the EBNA2 promoter. Competition of binding reactions was performed in the presence of unlabeled oligonucleotides containing homologous RBPJ consensus sequences in 200-fold excess. DNA-nuclear protein complexes were resolved by gel electrophoresis, transferred to a nylon membrane, cross-linked with UV-light, exposed to a streptavidin-horseradish peroxidase conjugate and visualized by chemiluminescence. The arrows indicate the position of the DNA/protein complexes.
    㠎 â 1 4 M Biotinylated Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Visualizing unwinding of individual DNA molecules using fluorescent SSB protein and TIRF microscopy. (A) Top : Diagram of phage λ DNA molecule with the indicated number of biotin groups incorporated in each 12-nt cos overhang. Middle : Illustration of biotinylated λ DNA attached at both ends via biotin–streptavidin linkage. Bottom : Image of an actual λ DNA molecule attached to the glass surface, stained with YO-PRO-1 (100 n M ), and illuminated with a 488 nm laser. The image is false colored in green and the attachment points to the glass surface are indicated. (B) The process required to construct a flow cell containing three separate single-channels. The steps highlighted are equivalent to those described in Section 4.1. (C) The flow cell from (B) mounted onto the objective; biotinylated lambda DNA was injected under buffer flow, permitting attachment of both ends to the surface. Unwinding tracks are visualized by binding of AF488-SSB G26C ( green ) to ssDNA regions. (D) Schematic representation of a TIRF microscope capable of visualizing DNA unwinding by RecQ by monitoring signal from both DNA and fluorescent SSB simultaneously. As shown in (C) the flow cell is mounted onto a 100× oil-immersion objective. The fluorescent SSB and DNA are excited by two lasers; 488 and 561 nm, respectively, and emission measured, via dichroic mirrors (M1 and M2). The deconvoluted emission is then directed onto different areas of a CCD camera generating a signal corresponding to either SSB or DNA. The lasers are operated using a custom LABview VI program to coordinate excitation with image acquisition so that the sample is illuminated only during the exposure times. Panels (A) and (D): From . Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding. Proceedings of the National Academy of Sciences of the United States of America, 112 (50), E6852 – E6861 .

    Journal: Methods in enzymology

    Article Title: Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases

    doi: 10.1016/bs.mie.2016.09.010

    Figure Lengend Snippet: Visualizing unwinding of individual DNA molecules using fluorescent SSB protein and TIRF microscopy. (A) Top : Diagram of phage λ DNA molecule with the indicated number of biotin groups incorporated in each 12-nt cos overhang. Middle : Illustration of biotinylated λ DNA attached at both ends via biotin–streptavidin linkage. Bottom : Image of an actual λ DNA molecule attached to the glass surface, stained with YO-PRO-1 (100 n M ), and illuminated with a 488 nm laser. The image is false colored in green and the attachment points to the glass surface are indicated. (B) The process required to construct a flow cell containing three separate single-channels. The steps highlighted are equivalent to those described in Section 4.1. (C) The flow cell from (B) mounted onto the objective; biotinylated lambda DNA was injected under buffer flow, permitting attachment of both ends to the surface. Unwinding tracks are visualized by binding of AF488-SSB G26C ( green ) to ssDNA regions. (D) Schematic representation of a TIRF microscope capable of visualizing DNA unwinding by RecQ by monitoring signal from both DNA and fluorescent SSB simultaneously. As shown in (C) the flow cell is mounted onto a 100× oil-immersion objective. The fluorescent SSB and DNA are excited by two lasers; 488 and 561 nm, respectively, and emission measured, via dichroic mirrors (M1 and M2). The deconvoluted emission is then directed onto different areas of a CCD camera generating a signal corresponding to either SSB or DNA. The lasers are operated using a custom LABview VI program to coordinate excitation with image acquisition so that the sample is illuminated only during the exposure times. Panels (A) and (D): From . Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding. Proceedings of the National Academy of Sciences of the United States of America, 112 (50), E6852 – E6861 .

    Article Snippet: Subsequently, inject 100 μL of 1 mg/mL biotinylated BSA (Thermo Scientific), in the same buffer (25 m M TrisOAc (pH 7.5), and 50 m M NaCl), to coat the flow cell.

    Techniques: Microscopy, Staining, Construct, Flow Cytometry, Lambda DNA Preparation, Injection, Binding Assay

    Three viral noncoding RNAs were identified and confirmed to be directly associated with K8 protein. (A) CLIP-seq reads mapped to the KSHV genome. Enlargement of the K8 cross-linked RNA in the KSHV genomic location showed two peaks (T1.4 RNA and PAN RNA) from the forward strain (left) and one peak (T0.7 RNA) from the reverse strain (right). (B and C) PAN (B) and T1.4 (C) RNAs and their truncation mutants were synthesized in vitro and labeled with biotin. An in vitro RNA pulldown assay was performed to evaluate the abilities of the RNAs and their mutants to be bound by K8. Biotinylated transcripts were mixed with purified GST-K8 protein, and the RNA-protein complexes were precipitated with streptavidin-coupled Dynabeads. The precipitates were analyzed by Western blotting for RNA-bound GST-K8 using anti-GST antibody and by Northern blotting for RNAs using the chemiluminescent nucleic acid detection module.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

    doi: 10.1128/JVI.02177-17

    Figure Lengend Snippet: Three viral noncoding RNAs were identified and confirmed to be directly associated with K8 protein. (A) CLIP-seq reads mapped to the KSHV genome. Enlargement of the K8 cross-linked RNA in the KSHV genomic location showed two peaks (T1.4 RNA and PAN RNA) from the forward strain (left) and one peak (T0.7 RNA) from the reverse strain (right). (B and C) PAN (B) and T1.4 (C) RNAs and their truncation mutants were synthesized in vitro and labeled with biotin. An in vitro RNA pulldown assay was performed to evaluate the abilities of the RNAs and their mutants to be bound by K8. Biotinylated transcripts were mixed with purified GST-K8 protein, and the RNA-protein complexes were precipitated with streptavidin-coupled Dynabeads. The precipitates were analyzed by Western blotting for RNA-bound GST-K8 using anti-GST antibody and by Northern blotting for RNAs using the chemiluminescent nucleic acid detection module.

    Article Snippet: Since T1.4 RNA contains high-GC-repeat sequence, biotinylated T1.4 was generated by adding biotin at the 3′ end of in vitro -transcribed T1.4 RNA with the Pierce biotin 3′ end DNA-labeling kit (Thermo Fisher).

    Techniques: Cross-linking Immunoprecipitation, Synthesized, In Vitro, Labeling, Purification, Western Blot, Northern Blot

    Mapping of K8 for the RNA binding domain. (A) Schematic presentation of K8 truncation, deletion, or amino acid substitution mutants that were used for mapping the RNA binding domain. The binding properties with T1.4, PAN, MRP, and 7SK RNAs are shown on the left of each construct. NLS, nuclear localization signal. GRXGR is the consensus RNA binding motif of the DEAD box protein. The symbols ++, +, and − represent strong, moderate, and no binding, respectively. (B) Expression vectors of Myc-K8 and mutants were introduced into 293T cells. Cell lysates were prepared and mixed with streptavidin beads coated with biotinylated RNA. The RNA pulldown materials were analyzed by Western blotting with an anti-Myc antibody. Shown are the results from the 7SK RNA pulldown assay. IB, immunoblotting. (C) Effects of GDDG mutation on binding of K8 with hnRNP U determined by immunoprecipitation with anti-Myc antibody and Western blotting using anti-Flag antibody.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

    doi: 10.1128/JVI.02177-17

    Figure Lengend Snippet: Mapping of K8 for the RNA binding domain. (A) Schematic presentation of K8 truncation, deletion, or amino acid substitution mutants that were used for mapping the RNA binding domain. The binding properties with T1.4, PAN, MRP, and 7SK RNAs are shown on the left of each construct. NLS, nuclear localization signal. GRXGR is the consensus RNA binding motif of the DEAD box protein. The symbols ++, +, and − represent strong, moderate, and no binding, respectively. (B) Expression vectors of Myc-K8 and mutants were introduced into 293T cells. Cell lysates were prepared and mixed with streptavidin beads coated with biotinylated RNA. The RNA pulldown materials were analyzed by Western blotting with an anti-Myc antibody. Shown are the results from the 7SK RNA pulldown assay. IB, immunoblotting. (C) Effects of GDDG mutation on binding of K8 with hnRNP U determined by immunoprecipitation with anti-Myc antibody and Western blotting using anti-Flag antibody.

    Article Snippet: Since T1.4 RNA contains high-GC-repeat sequence, biotinylated T1.4 was generated by adding biotin at the 3′ end of in vitro -transcribed T1.4 RNA with the Pierce biotin 3′ end DNA-labeling kit (Thermo Fisher).

    Techniques: RNA Binding Assay, Binding Assay, Construct, Expressing, Western Blot, Mutagenesis, Immunoprecipitation

    T1.4 RNA associated with ori-Lyt DNA. (A) Alignment of T1.4 RNA with ori-Lyt DNA sequences in the KSHV genome revealing potential base pairing between them. (B) Schematic illustration of the ChIRP assay procedure. Glutaraldehyde was applied to induce DNA-RNA cross-linking. Chromatin was sonicated and then incubated with biotinylated oligonucleotide probe specifically to the target RNA. Streptavidin beads were added to pull down the biotinylated RNA and bound DNA. qPCR was used to detect the ori-Lyt DNA. (C) Validation of the efficiency of T1.4 probe in pulldown of T1.4 RNA. A ChIRP assay was performed using probes targeting T1.4 or LacZ. Precipitated RNAs were purified and subjected to RT-qPCR with primer against T1.4. (D) T1.4 RNA associates with ori-Lyt DNA. Two sets of probes were used to perform ChIRP experiments. RNase H, RNase A, or trypsin was added in washing buffer during the experiments to digest the RNA or protein. The pulled down DNA was amplified by primers for the 12F region of ori-Lyt. The error bars indicate SD.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus K8 Is an RNA Binding Protein That Regulates Viral DNA Replication in Coordination with a Noncoding RNA

    doi: 10.1128/JVI.02177-17

    Figure Lengend Snippet: T1.4 RNA associated with ori-Lyt DNA. (A) Alignment of T1.4 RNA with ori-Lyt DNA sequences in the KSHV genome revealing potential base pairing between them. (B) Schematic illustration of the ChIRP assay procedure. Glutaraldehyde was applied to induce DNA-RNA cross-linking. Chromatin was sonicated and then incubated with biotinylated oligonucleotide probe specifically to the target RNA. Streptavidin beads were added to pull down the biotinylated RNA and bound DNA. qPCR was used to detect the ori-Lyt DNA. (C) Validation of the efficiency of T1.4 probe in pulldown of T1.4 RNA. A ChIRP assay was performed using probes targeting T1.4 or LacZ. Precipitated RNAs were purified and subjected to RT-qPCR with primer against T1.4. (D) T1.4 RNA associates with ori-Lyt DNA. Two sets of probes were used to perform ChIRP experiments. RNase H, RNase A, or trypsin was added in washing buffer during the experiments to digest the RNA or protein. The pulled down DNA was amplified by primers for the 12F region of ori-Lyt. The error bars indicate SD.

    Article Snippet: Since T1.4 RNA contains high-GC-repeat sequence, biotinylated T1.4 was generated by adding biotin at the 3′ end of in vitro -transcribed T1.4 RNA with the Pierce biotin 3′ end DNA-labeling kit (Thermo Fisher).

    Techniques: Sonication, Incubation, Real-time Polymerase Chain Reaction, Purification, Quantitative RT-PCR, Amplification

    Cortisol has a modest impact on RBPJ/DNA interactions. Primary osteoblast-enriched cells were seeded on BSA or DLL1, confluent cultures maintained for 24 h in the absence of serum and then exposed to vehicle or cortisol 1 μM for 6 h. Nuclear proteins were extracted and binding reactions carried out with a biotinylated oligonucleotide containing an RBPJ consensus sequence from the EBNA2 promoter. Competition of binding reactions was performed in the presence of unlabeled oligonucleotides containing homologous RBPJ consensus sequences in 200-fold excess. DNA-nuclear protein complexes were resolved by gel electrophoresis, transferred to a nylon membrane, cross-linked with UV-light, exposed to a streptavidin-horseradish peroxidase conjugate and visualized by chemiluminescence. The arrows indicate the position of the DNA/protein complexes.

    Journal: Journal of cellular biochemistry

    Article Title: GLUCOCORTICOIDS INHIBIT NOTCH TARGET GENE EXPRESSION IN OSTEOBLASTS

    doi: 10.1002/jcb.26798

    Figure Lengend Snippet: Cortisol has a modest impact on RBPJ/DNA interactions. Primary osteoblast-enriched cells were seeded on BSA or DLL1, confluent cultures maintained for 24 h in the absence of serum and then exposed to vehicle or cortisol 1 μM for 6 h. Nuclear proteins were extracted and binding reactions carried out with a biotinylated oligonucleotide containing an RBPJ consensus sequence from the EBNA2 promoter. Competition of binding reactions was performed in the presence of unlabeled oligonucleotides containing homologous RBPJ consensus sequences in 200-fold excess. DNA-nuclear protein complexes were resolved by gel electrophoresis, transferred to a nylon membrane, cross-linked with UV-light, exposed to a streptavidin-horseradish peroxidase conjugate and visualized by chemiluminescence. The arrows indicate the position of the DNA/protein complexes.

    Article Snippet: Binding reactions of 1 μM biotinylated DNA with 5 μg of nuclear extracts were carried out with the LightShift Chemiluminescent EMSA Kit, as recommended by the manufacturer (Thermo Fisher Scientific).

    Techniques: Binding Assay, Sequencing, Nucleic Acid Electrophoresis

    Menin binds to E-box through interacting with MYC. ( a , b ) HEK293T cells were transfected with Flag-Menin or HA-MYC or both vectors. IP was performed with anti-Flag ( a ) or anti-HA ( b ), followed by immunoblot analysis. M: protein marker. ( c ) GST pull-down assay was performed with GST or GST fused MYC protein and His-Menin protein. ( d ) Endogenous MYC and Menin interaction. IP of endogenous Menin, MYC and MAX proteins from 293T cells lysed in high-salt buffer. IP was performed with anti-IgG or anti-Mein antibody. ( e ) GST or GST fused MYC truncated proteins TAD, CP and bHLHZ were purified (on the bottom) and used for GST pull-down assay with His-Menin. FL, full length. ( f ) GST pull-down assay was performed with GST or GST fused MAX protein and His-MYC (left panel) or His-Menin (right panel). ( g ) EMSA assays of purified MAX/MAX, MYC/MAX or Menin binding to biotinylated canonical CACGTG E-box sequences following competition with decreasing amounts (20-, 10-, 5-, 2- and 1-fold excess) of unlabelled competitor sequences. The higher bands reflect the amounts of labelled E-box bound by MYC/MAX or MAX/MAX and the lower band reflects the amount of unbound E-box DNA. Lane 1: no proteins. Lane 2: MAX/MAX with no competitor DNA. Lane 3: MYC/MAX with no competitor DNA. Lanes 4–8: the effect of adding indicated amounts of competitor DNA fragments containing the canonical E-box. Lane 9: Menin with no competitor DNA. Bottom: free biotinylated DNA. ( h ) EMSA assay analysing the binding of MAX/MAX, MYC/MAX or MYC/MAX/Menin to biotinylated canonical CACGTG E-box sequences in the presence or absence of anti-Menin antibody.

    Journal: Nature Communications

    Article Title: Menin enhances c-Myc-mediated transcription to promote cancer progression

    doi: 10.1038/ncomms15278

    Figure Lengend Snippet: Menin binds to E-box through interacting with MYC. ( a , b ) HEK293T cells were transfected with Flag-Menin or HA-MYC or both vectors. IP was performed with anti-Flag ( a ) or anti-HA ( b ), followed by immunoblot analysis. M: protein marker. ( c ) GST pull-down assay was performed with GST or GST fused MYC protein and His-Menin protein. ( d ) Endogenous MYC and Menin interaction. IP of endogenous Menin, MYC and MAX proteins from 293T cells lysed in high-salt buffer. IP was performed with anti-IgG or anti-Mein antibody. ( e ) GST or GST fused MYC truncated proteins TAD, CP and bHLHZ were purified (on the bottom) and used for GST pull-down assay with His-Menin. FL, full length. ( f ) GST pull-down assay was performed with GST or GST fused MAX protein and His-MYC (left panel) or His-Menin (right panel). ( g ) EMSA assays of purified MAX/MAX, MYC/MAX or Menin binding to biotinylated canonical CACGTG E-box sequences following competition with decreasing amounts (20-, 10-, 5-, 2- and 1-fold excess) of unlabelled competitor sequences. The higher bands reflect the amounts of labelled E-box bound by MYC/MAX or MAX/MAX and the lower band reflects the amount of unbound E-box DNA. Lane 1: no proteins. Lane 2: MAX/MAX with no competitor DNA. Lane 3: MYC/MAX with no competitor DNA. Lanes 4–8: the effect of adding indicated amounts of competitor DNA fragments containing the canonical E-box. Lane 9: Menin with no competitor DNA. Bottom: free biotinylated DNA. ( h ) EMSA assay analysing the binding of MAX/MAX, MYC/MAX or MYC/MAX/Menin to biotinylated canonical CACGTG E-box sequences in the presence or absence of anti-Menin antibody.

    Article Snippet: DNA was UV crosslinked to membrane and biotinylated DNA was detected using Pierce Chemiluminescent Nucleic Acid Detection Module (Thermo).

    Techniques: Transfection, Marker, Pull Down Assay, Purification, Binding Assay