luciferase assay Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Millipore luciferase
    Luciferase, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 763 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase/product/Millipore
    Average 93 stars, based on 763 article reviews
    Price from $9.99 to $1999.99
    luciferase - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    99
    Millipore luciferase assay
    Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing <t>shRNA</t> against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) <t>shLuc-</t> (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p
    Luciferase Assay, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase assay/product/Millipore
    Average 99 stars, based on 595 article reviews
    Price from $9.99 to $1999.99
    luciferase assay - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    90
    Millipore luciferase enzyme
    Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing <t>shRNA</t> against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) <t>shLuc-</t> (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p
    Luciferase Enzyme, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase enzyme/product/Millipore
    Average 90 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    luciferase enzyme - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    94
    Millipore anti luciferase
    Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing <t>shRNA</t> against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) <t>shLuc-</t> (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p
    Anti Luciferase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti luciferase/product/Millipore
    Average 94 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    anti luciferase - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    Image Search Results


    Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing shRNA against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) shLuc- (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p

    Journal: PLoS ONE

    Article Title: Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages

    doi: 10.1371/journal.pone.0145617

    Figure Lengend Snippet: Phagocytosis activity in PLSCR1-depleted and -overexpressing cells. (A-C) THP-1 cells were transduced with lentiviruses expressing shRNA against PLSCR1 or Luciferase, treated with PMA for 72 h as indicated in Fig 4 , and then submitted to phagocytosis of IgG-opsonised SRBCs. (A) shLuc- (upper panels) or shPLSCR1-transduced (lower panels) cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa350-anti-rabbit IgG, anti-PLSCR1 (1E9 mAb) revealed with Alexa555-anti-mouse IgG, and Alexa488-phalloidin to reveal internalized SRBCs, PLSCR1 and F-actin, respectively. (B and C) shLuc- or shPLSCR1-transduced cells were incubated for 2, 5, 10 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-rabbit IgG. The number of external SRBCs and the total number of cell-associated SRBCs in each cell was counted for at least 100 cells in each experiment. In (B), results are expressed as the percentage of cell-associated (internal and external) SRBCs/cell relative to that measured in shLuc-transduced cells after 30 min of phagocytosis. In (C), results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis in shLuc-transduced control cells. (D and E) THP-1 cells were transfected with the vector for expression of HA-PLSCR1, and 18 hours later tested for phagocytosis of IgG-opsonised SRBCs. In (D), transfected cells were incubated for 30 min at 37°C with opsonised SRBCs, then fixed, permeabilized and stained with Alexa647-anti-rabbit IgG, anti-HA rat antibody revealed with Alexa488-anti-rat IgG, and Alexa555-phalloidin to reveal internalized SRBCs, HA-PLSCR1 and F-actin, respectively. In (E), cells were incubated for 5, 15 or 30 min at 37°C with opsonised SRBCs, then fixed and stained with Alexa647-anti-rabbit IgG to detect external SRBCs before permeabilization for labeling of all cell-associated SRBCs including internalized particles with Alexa350-anti-Rabbit. Cells were also stained with anti-HA rat antibody revealed with Alexa488-anti-rat IgG to identify transfected cells. The number of external SRBCs and the total number of cell-associated SRBCs in each cells were counted for at least 50 transfected cells and 50 non-transfected cells. Results are expressed as the percentage of phagocytosed SRBCs/cell relative to that measured after 30 min of phagocytosis for non-transfected control cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using students t test (n.s., p > 0.05; *, p

    Article Snippet: The pLKO.1 plasmid expressing shRNA against firefly luciferase (shLuc) (Sigma) was used to produce control particles.

    Techniques: Activity Assay, Transduction, Expressing, shRNA, Luciferase, Incubation, Staining, Labeling, Transfection, Plasmid Preparation

    Monocyte-to-macrophage differentiation and cell surface phosphatidylserine exposure in PLSCR1-depleted cells. THP-1 cells were transduced with lentiviruses expressing shRNA against either PLSCR1 or Luciferase used as a control, and then cultured for 72 h with or without PMA as previously. (A) Lysates from shRNA-transduced THP-1 cells were analyzed by Western blotting with anti-PLSCR1 (upper panels) and anti-ɣ-tubulin (lower panels). (B and C) Cell surface expression of CD14 and PS exposure. Treated or untreated shRNA-transduced THP-1 cells were stained with FITC-conjugated anti-CD14 antibodies (B) or PE-conjugated Annexin-V (C), and surface expression was measured by flow cytometry. Results are expressed as the percentage of the MFI relative to the non differentiated control shLuc-transduced cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using Student's t test (non significant, p > 0.05).

    Journal: PLoS ONE

    Article Title: Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages

    doi: 10.1371/journal.pone.0145617

    Figure Lengend Snippet: Monocyte-to-macrophage differentiation and cell surface phosphatidylserine exposure in PLSCR1-depleted cells. THP-1 cells were transduced with lentiviruses expressing shRNA against either PLSCR1 or Luciferase used as a control, and then cultured for 72 h with or without PMA as previously. (A) Lysates from shRNA-transduced THP-1 cells were analyzed by Western blotting with anti-PLSCR1 (upper panels) and anti-ɣ-tubulin (lower panels). (B and C) Cell surface expression of CD14 and PS exposure. Treated or untreated shRNA-transduced THP-1 cells were stained with FITC-conjugated anti-CD14 antibodies (B) or PE-conjugated Annexin-V (C), and surface expression was measured by flow cytometry. Results are expressed as the percentage of the MFI relative to the non differentiated control shLuc-transduced cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using Student's t test (non significant, p > 0.05).

    Article Snippet: The pLKO.1 plasmid expressing shRNA against firefly luciferase (shLuc) (Sigma) was used to produce control particles.

    Techniques: Transduction, Expressing, shRNA, Luciferase, Cell Culture, Western Blot, Staining, Flow Cytometry, Cytometry

    Regulation of the Wnt/β-catenin pathway by the α6A variant subunit. Representative WB and graph of the densiometric analysis for the detection of active β-catenin and total β-catenin in the whole cell extract ( A ) and β-catenin in nuclear extracts ( B ). β-Actin served as loading control in cell extracts and histone H1 for nuclear extracts. Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3. ( C ) TOPflash assay of the response of β-catenin/TCF4 promotor activity in the α6A variant knocked down cell lines and their corresponding shctrl. Results showed the net luciferase/renilla ratio (Topflash − FOPflash). Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3.

    Journal: Carcinogenesis

    Article Title: Integrin ?6A splice variant regulates proliferation and the Wnt/?-catenin pathway in human colorectal cancer cells

    doi: 10.1093/carcin/bgu006

    Figure Lengend Snippet: Regulation of the Wnt/β-catenin pathway by the α6A variant subunit. Representative WB and graph of the densiometric analysis for the detection of active β-catenin and total β-catenin in the whole cell extract ( A ) and β-catenin in nuclear extracts ( B ). β-Actin served as loading control in cell extracts and histone H1 for nuclear extracts. Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3. ( C ) TOPflash assay of the response of β-catenin/TCF4 promotor activity in the α6A variant knocked down cell lines and their corresponding shctrl. Results showed the net luciferase/renilla ratio (Topflash − FOPflash). Statistical analysis between shctrl and shα6A: * P ≤ 0.05, ** P ≤ 0.01, t -test, n = 3.

    Article Snippet: Transfections and luciferase assays TOPflash and FOPflash reporter plasmids (Millipore) were transfected into CRC cell lines with Effectene transfection reagent (Qiagen) using the manufacturer’s instructions.

    Techniques: Variant Assay, Western Blot, TOPFlash assay, Activity Assay, Luciferase

    Biochemical assays performed on purified HP0231 and HP0231m. Purified EcDsbA, EcDsbC, or EcDsbG were used as controls. (A) HP0231m is not active in the insulin reduction assay. The reaction contained 131 μM insulin in potassium phosphate buffer, pH 7.0 and 2 mM EDTA. The reaction was performed in the absence or presence of 10 μM EcDsbA and 10 μM HP0231m. Reactions were started by adding DTT to the final concentration of 1 mM. Changes in the absorbance at 650 nm as a function of time were measured. The figure presents the average of three independent experiments ( n = 3). (B) HP0231 and HP0231m are active in an oxidase activity assay (reduced unfolded – ruRNase activity assay). Reactions were carried out in 200 μl of PBS buffer containing 100 mM Tris acetate pH 8.0, 2 mM EDTA, 0.2 mM GSSG, 1 mM GSH, 4,5 mM cCMP, ruRNaseA (10 μM) and the analyzed enzyme (20 μM). The reaction was performed in the absence or presence of 20 μM EcDsbA, 20 μM HP0231, or 20 μM HP0231m. Changes in absorbance at 296 nm as a function of time were measured. Three independent experiments were performed. The figure presents a representative result. (C) HP0231 and HP0231m cannot work as isomerases in the scrambled RNase (scRNase) activity assay. Reactions were carried out in 200 μl of PBS buffer containing 100 mM Tris acetate pH 8.0, 2 mM EDTA, 10 μM DTT, 4.5 mM cCMP, scRNaseA (40 μM) and the analyzed enzyme (20 μM). Reactions were performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0231, or 20 μM HP0231m. Changes in absorbance at 296 nm as a function of time were measured. Three independent experiments were performed. The figure presents a representative result. (D) HP0231 and HP0231m suppress the thermal aggregation of luciferase (LUC) in the chaperone activity assay. LUC was diluted to a final concentration of 0.10 μM into 40 mM HEPES-KOH buffer, pH 7.5, equilibrated at 43°C in the absence or in the presence of 0.15 μM HP0231 or 0.15 μM HP0231m, respectively. Protein aggregation was monitored with light scattering measurements using a Varian spectrofluorometer. The excitation and emission wavelengths were set to 350 nm. The excitation and emission slit widths were set to 2.5 nm. To exclude unspecific protein effects, control experiments in the presence of 1.5 μM bovine serum albumin were conducted. Three independent experiments were performed. The figure presents a representative result. (E) HP0231 and HP0231m suppress the thermal aggregation of citrate synthase (CS) at 43°C. 30 μM CS was diluted 200-fold into prewarmed 40 mM HEPES-KOH, pH 7.5, at 43°C in the absence or presence of 0.15 μM HP0231 and 1.5 μM HP0231m, respectively. Protein aggregation was monitored with light scattering measurements using a Varian spectrofluorometer. The excitation and emission wavelengths were set to 350 nm. The excitation and emission slit widths were set to 2.5 nm. To exclude non-specific protein effects, control experiments in the presence of 1.5 μM bovine serum albumin were conducted. Three independent experiments were performed. The figure presents a representative result.

    Journal: Frontiers in Microbiology

    Article Title: Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

    doi: 10.3389/fmicb.2015.01065

    Figure Lengend Snippet: Biochemical assays performed on purified HP0231 and HP0231m. Purified EcDsbA, EcDsbC, or EcDsbG were used as controls. (A) HP0231m is not active in the insulin reduction assay. The reaction contained 131 μM insulin in potassium phosphate buffer, pH 7.0 and 2 mM EDTA. The reaction was performed in the absence or presence of 10 μM EcDsbA and 10 μM HP0231m. Reactions were started by adding DTT to the final concentration of 1 mM. Changes in the absorbance at 650 nm as a function of time were measured. The figure presents the average of three independent experiments ( n = 3). (B) HP0231 and HP0231m are active in an oxidase activity assay (reduced unfolded – ruRNase activity assay). Reactions were carried out in 200 μl of PBS buffer containing 100 mM Tris acetate pH 8.0, 2 mM EDTA, 0.2 mM GSSG, 1 mM GSH, 4,5 mM cCMP, ruRNaseA (10 μM) and the analyzed enzyme (20 μM). The reaction was performed in the absence or presence of 20 μM EcDsbA, 20 μM HP0231, or 20 μM HP0231m. Changes in absorbance at 296 nm as a function of time were measured. Three independent experiments were performed. The figure presents a representative result. (C) HP0231 and HP0231m cannot work as isomerases in the scrambled RNase (scRNase) activity assay. Reactions were carried out in 200 μl of PBS buffer containing 100 mM Tris acetate pH 8.0, 2 mM EDTA, 10 μM DTT, 4.5 mM cCMP, scRNaseA (40 μM) and the analyzed enzyme (20 μM). Reactions were performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0231, or 20 μM HP0231m. Changes in absorbance at 296 nm as a function of time were measured. Three independent experiments were performed. The figure presents a representative result. (D) HP0231 and HP0231m suppress the thermal aggregation of luciferase (LUC) in the chaperone activity assay. LUC was diluted to a final concentration of 0.10 μM into 40 mM HEPES-KOH buffer, pH 7.5, equilibrated at 43°C in the absence or in the presence of 0.15 μM HP0231 or 0.15 μM HP0231m, respectively. Protein aggregation was monitored with light scattering measurements using a Varian spectrofluorometer. The excitation and emission wavelengths were set to 350 nm. The excitation and emission slit widths were set to 2.5 nm. To exclude unspecific protein effects, control experiments in the presence of 1.5 μM bovine serum albumin were conducted. Three independent experiments were performed. The figure presents a representative result. (E) HP0231 and HP0231m suppress the thermal aggregation of citrate synthase (CS) at 43°C. 30 μM CS was diluted 200-fold into prewarmed 40 mM HEPES-KOH, pH 7.5, at 43°C in the absence or presence of 0.15 μM HP0231 and 1.5 μM HP0231m, respectively. Protein aggregation was monitored with light scattering measurements using a Varian spectrofluorometer. The excitation and emission wavelengths were set to 350 nm. The excitation and emission slit widths were set to 2.5 nm. To exclude non-specific protein effects, control experiments in the presence of 1.5 μM bovine serum albumin were conducted. Three independent experiments were performed. The figure presents a representative result.

    Article Snippet: Chaperone Activity of HP0231 and HP0231m The chaperone activity of HP0231 and HP0231m, in comparison to EcDsbG, was determined as described previously ( ) using thermal aggregation of citrate synthase – CS (Sigma) and luciferase – LUC (Sigma) as chaperone substrate proteins.

    Techniques: Purification, Insulin Reduction Assay, Concentration Assay, Activity Assay, Luciferase