Journal: Microorganisms

Article Title: A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum

doi: 10.3390/microorganisms11010155

Figure Lengend Snippet: SDS-DNA PAGE of IF cells of H. obtusa. Calf thymus DNA was used. ( a ) CBB stained gel; ( b ) immunoblot with mAb3D1B9C4 specific for PRP1; ( c ) EB stained gel. Arrows, PRP1. Arrowheads, other EB-staining negative bands showing positions of DNA-protein complexes (see ).

Article Snippet: For SDS–DNA PAGE, a 10% ( w / v ) separation gel containing 10 μg mL –1 calf thymus DNA (Worthington Biochemical Corp., Lakewood, NJ, USA) or 10 μg mL –1 P. caudatum whole genome DNA was used.

Techniques: IF-cells, Staining, Western Blot

Journal: Microorganisms

Article Title: A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum

doi: 10.3390/microorganisms11010155

Figure Lengend Snippet: SDS-PAGE and immunoblots of fractions of DNA affinity column chromatography. Calf thymus DNA was used. ( a ) Fractions 1–10 eluted with Na, K-PB; ( b ) fractions 1′–10′ eluted with Na, K-PB containing 0.2 M NaCl. Upper, silver-stained gel. Lower, immunoblot with mAb3D1B9C4. ( c ) High magnification of a fraction number 3′ using silver staining. Not only PRP1 but also several silver staining bands were also confirmed. ( d ) CBB stained gel of IF cells of H. obtusa . Arrow, PRP1. Arrowheads, other bands eluted. Bands in ( c ) match the bands in ( d ).

Article Snippet: For SDS–DNA PAGE, a 10% ( w / v ) separation gel containing 10 μg mL –1 calf thymus DNA (Worthington Biochemical Corp., Lakewood, NJ, USA) or 10 μg mL –1 P. caudatum whole genome DNA was used.

Techniques: SDS Page, Western Blot, Affinity Column, Chromatography, Staining, Silver Staining, IF-cells

Journal: Microorganisms

Article Title: A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum

doi: 10.3390/microorganisms11010155

Figure Lengend Snippet: SDS-DNA PAGE of IF cells of H. obtusa . P. caudatum DNA was used. ( a ) CBB stained gel. ( b ) Immunoblot with mAb3D1B9C4 specific for PRP1. ( c ) EB stained gel. Arrows, 63-kDa PRP1. It should be noted that not only PRP1 but also many EB-negative bands that show the positions of DNA-protein complexes appeared (see ).

Article Snippet: For SDS–DNA PAGE, a 10% ( w / v ) separation gel containing 10 μg mL –1 calf thymus DNA (Worthington Biochemical Corp., Lakewood, NJ, USA) or 10 μg mL –1 P. caudatum whole genome DNA was used.

Techniques: IF-cells, Staining, Western Blot

Journal: Microorganisms

Article Title: A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum

doi: 10.3390/microorganisms11010155

Figure Lengend Snippet: DNA affinity chromatography of H. obtusa IFs. P. caudatum DNA was used. ( a ) Silver stained SDS-PAGE gel; ( b ) immunoblotting with the monoclonal antibody mAb3D1B9C4 specific for the 63-kDa periplasmic protein PRP1 of H. obtusa . Lane 1, supernatant of sonicated H. obtusa before applying to DNA affinity chromatography column; lane 2, first elute of supernatant of sonicated H. obtusa after applying to the column; lane 3, first elute by elution buffer containing 0.02 M NaCl; lane 4, first eluate by 0.1 M NaCl elution buffer; lane 5, first eluate by 0.2 M NaCl elution buffer; lane 6, first eluate by 0.3 M NaCl elution buffer; lane 7, first eluate by 2 M NaCl elution buffer; arrows, PRP1. Eluates from each column were collected in every 200 μL, and the first eluate from each column was used for SDS-PAGE and immunoblot with mAb. Note that not only PRP1 but also several silver stained bands were confirmed in lane 4 of ( a ).

Article Snippet: For SDS–DNA PAGE, a 10% ( w / v ) separation gel containing 10 μg mL –1 calf thymus DNA (Worthington Biochemical Corp., Lakewood, NJ, USA) or 10 μg mL –1 P. caudatum whole genome DNA was used.

Techniques: Affinity Chromatography, Staining, SDS Page, Western Blot, Sonication, Affinity Column

Journal: Microorganisms

Article Title: A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum

doi: 10.3390/microorganisms11010155

Figure Lengend Snippet: SDS-DNA PAGE of IF cells of H. obtusa . P. caudatum DNA was used. ( a ) CBB stained gel. ( b ) Immunoblot with mAb3D1B9C4 specific for PRP1. ( c ) EB stained gel. Arrows, 63-kDa PRP1. It should be noted that not only PRP1 but also many EB-negative bands that show the positions of DNA-protein complexes appeared (see ).

Article Snippet: For SDS–DNA PAGE, a 10% ( w / v ) separation gel containing 10 μg mL –1 calf thymus DNA (Worthington Biochemical Corp., Lakewood, NJ, USA) or 10 μg mL –1 P. caudatum whole genome DNA was used.

Techniques: IF-cells, Staining, Western Blot

Journal: Microorganisms

Article Title: A 63-kDa Periplasmic Protein of the Endonuclear Symbiotic Bacterium Holospora obtusa Secreted to the Outside of the Bacterium during the Early Infection Process Binds Weakly to the Macronuclear DNA of the Host Paramecium caudatum

doi: 10.3390/microorganisms11010155

Figure Lengend Snippet: DNA affinity chromatography of H. obtusa IFs. P. caudatum DNA was used. ( a ) Silver stained SDS-PAGE gel; ( b ) immunoblotting with the monoclonal antibody mAb3D1B9C4 specific for the 63-kDa periplasmic protein PRP1 of H. obtusa . Lane 1, supernatant of sonicated H. obtusa before applying to DNA affinity chromatography column; lane 2, first elute of supernatant of sonicated H. obtusa after applying to the column; lane 3, first elute by elution buffer containing 0.02 M NaCl; lane 4, first eluate by 0.1 M NaCl elution buffer; lane 5, first eluate by 0.2 M NaCl elution buffer; lane 6, first eluate by 0.3 M NaCl elution buffer; lane 7, first eluate by 2 M NaCl elution buffer; arrows, PRP1. Eluates from each column were collected in every 200 μL, and the first eluate from each column was used for SDS-PAGE and immunoblot with mAb. Note that not only PRP1 but also several silver stained bands were confirmed in lane 4 of ( a ).

Article Snippet: For SDS–DNA PAGE, a 10% ( w / v ) separation gel containing 10 μg mL –1 calf thymus DNA (Worthington Biochemical Corp., Lakewood, NJ, USA) or 10 μg mL –1 P. caudatum whole genome DNA was used.

Techniques: Affinity Chromatography, Staining, SDS Page, Western Blot, Sonication, Affinity Column

Journal: PLoS ONE

Article Title: The Inhibition of Anti-DNA Binding to DNA by Nucleic Acid Binding Polymers

doi: 10.1371/journal.pone.0040862

Figure Lengend Snippet: The ability of polymers PAMAM, HDMBr, and CDP to inhibit binding of a murine monoclonal anti-DNA antibody QB1 to DNA was tested by ELISA. Binding of QB1 at 35 ng/ml final concentration was assayed by ELISA in the presence of PAMAM, HDMBr, or CDP at final concentrations from 10 ng/ml to 1,250 ng/ml. Control wells had buffer alone. Antibody was measured as described in Experimental Procedures. The OD 450 values of 3 wells of each polymer concentration or of 6 wells without polymer were averaged. Each point shown is 100% x average OD 450 with polymer/average OD 450 without polymer. Circles show data for PAMAM; squares show data for HDMBr; triangles show data for CDP.

Article Snippet: For the direct binding assay, wells of ELISA plates were coated with 5 µg/ml native double stranded calf thymus (CT) DNA (Worthington Biochemical, Lakewood, NJ) in 1 x SSC (150 mM NaCl, 15 mM Na citrate, pH 7) overnight at 4°C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: PLoS ONE

Article Title: The Inhibition of Anti-DNA Binding to DNA by Nucleic Acid Binding Polymers

doi: 10.1371/journal.pone.0040862

Figure Lengend Snippet: The ability of polymers PAMAM, HDMBr, and CDP to inhibit DNA binding by anti-DNA antibodies in three SLE patient plasmas to DNA was tested by ELISA. Binding by antibodies in Plasma 1 (final dilution 1/1,000), Plasma 2 (final dilution 1/2,800), and Plasma 3 (final dilution 1/3,000) was assayed in the presence of PAMAM, HDMBr, or CDP at final concentrations ranging from 300 ng/ml to 10,000 ng/ml or with dilution buffer alone. Antibody levels were determined by ELISA as described in . The OD 450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD 450 with polymer/average OD 450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3.

Article Snippet: For the direct binding assay, wells of ELISA plates were coated with 5 µg/ml native double stranded calf thymus (CT) DNA (Worthington Biochemical, Lakewood, NJ) in 1 x SSC (150 mM NaCl, 15 mM Na citrate, pH 7) overnight at 4°C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Inhibition of Anti-DNA Binding to DNA by Nucleic Acid Binding Polymers

doi: 10.1371/journal.pone.0040862

Figure Lengend Snippet: Native CT DNA was biotinylated and then captured in ELISA plate wells coated with streptavidin. The binding of Plasma 1 (final dilution 1/3,500), Plasma 2 (final dilution 1/8,000), and Plasma 3 (final dilution 1/4,500) was measured by ELISA in the presence of PAMAM, HDMBr, or CDP at final concentrations from 80 ng/ml to 10,000 ng/ml or with dilution buffer alone. The OD 450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD 450 with polymer/average OD 450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3.

Article Snippet: For the direct binding assay, wells of ELISA plates were coated with 5 µg/ml native double stranded calf thymus (CT) DNA (Worthington Biochemical, Lakewood, NJ) in 1 x SSC (150 mM NaCl, 15 mM Na citrate, pH 7) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: PLoS ONE

Article Title: The Inhibition of Anti-DNA Binding to DNA by Nucleic Acid Binding Polymers

doi: 10.1371/journal.pone.0040862

Figure Lengend Snippet: In this assay, 50 µl/well of Plasma 1 diluted 1/1,700, Plasma 2 diluted 1/3,950, and Plasma 3 diluted 1/2,250 were incubated in wells of microtiter plates with biotinylated DNA bound to streptavidin. After 1 hour to allow the formation of immune complexes, 50 µl of dilutions of PAMAM, HDMBr, or CDP or 50 µl of dilution buffer alone were then added to produce concentrations of the polymers of 10,000 ng/ml, 2,500 ng/ml, 620 ng/ml, 160 ng/ml or 0 ng/ml. Antibody binding was then determined by ELISA. The OD 450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD 450 with polymer/average OD 450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3.

Article Snippet: For the direct binding assay, wells of ELISA plates were coated with 5 µg/ml native double stranded calf thymus (CT) DNA (Worthington Biochemical, Lakewood, NJ) in 1 x SSC (150 mM NaCl, 15 mM Na citrate, pH 7) overnight at 4°C.

Techniques: Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay