elisa  (Sino Biological)


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    Name:
    Transferrin Receptor TFRC Protein Mouse Recombinant
    Description:
    A DNA sequence encoding the extracellular domain of mouse TFRC Q62351 Cys 89 Phe 763 was fused with a polyhistidine tag at the N terminus
    Catalog Number:
    50741-M07H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    2610028K12Rik Protein Mouse, AI195355 Protein Mouse, AI426448 Protein Mouse, AU015758 Protein Mouse, CD71 Protein Mouse, E430033M20Rik Protein Mouse, Mtvr-1 Protein Mouse, Mtvr1 Protein Mouse, p90 Protein Mouse, TFR Protein Mouse, TFR1 Protein Mouse, TR Protein Mouse, Trfr Protein Mouse
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological elisa
    A DNA sequence encoding the extracellular domain of mouse TFRC Q62351 Cys 89 Phe 763 was fused with a polyhistidine tag at the N terminus
    https://www.bioz.com/result/elisa/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    elisa - by Bioz Stars, 2021-07
    94/100 stars

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    Related Articles

    other:

    Article Title: Identification and in vivo characterization of a brain-penetrating nanobody
    Article Snippet: Mouse transferrin receptor

    Concentration Assay:

    Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model
    Article Snippet: Plasma Antibody QuantificationFor plasma concentration determination, standard 96-well sector plates (L15XA-1, Meso Scale Discovery) were coated with 0.5 μg/mL of purified extracellular domain of mTfR (50741-M07H, Sinobiological) or 0.5 μg/mL of purified human Aβ peptide (H-1368, Bachem) in PBS and then incubated for 1 h under agitation at room temperature. .. Plasma Antibody QuantificationFor plasma concentration determination, standard 96-well sector plates (L15XA-1, Meso Scale Discovery) were coated with 0.5 μg/mL of purified extracellular domain of mTfR (50741-M07H, Sinobiological) or 0.5 μg/mL of purified human Aβ peptide (H-1368, Bachem) in PBS and then incubated for 1 h under agitation at room temperature. .. After incubation, the plates were washed three times with PBS-Tween 0.05% (524653, Calbiochem) and blocked for 1 h at room temperature with 0.1% BSA solution (A7030, Sigma).

    Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model
    Article Snippet: In such a kinetic analysis, anti-mouse IgG Fc capture (AMC) biosensors (18-5088, ForteBio) were incubated with variant antibodies diluted in DPBS 1× at 50 nM. .. Then the biosensors were soaked in a concentration range of mTfR (50741-M07H, Sino Biological) for 300 s and the association rate was measured. .. Raw data analysis, including blank subtraction, fitting, and kinetic parameters calculations, were performed with ForteBio Data Analysis 10.0 software.

    Purification:

    Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model
    Article Snippet: Plasma Antibody QuantificationFor plasma concentration determination, standard 96-well sector plates (L15XA-1, Meso Scale Discovery) were coated with 0.5 μg/mL of purified extracellular domain of mTfR (50741-M07H, Sinobiological) or 0.5 μg/mL of purified human Aβ peptide (H-1368, Bachem) in PBS and then incubated for 1 h under agitation at room temperature. .. Plasma Antibody QuantificationFor plasma concentration determination, standard 96-well sector plates (L15XA-1, Meso Scale Discovery) were coated with 0.5 μg/mL of purified extracellular domain of mTfR (50741-M07H, Sinobiological) or 0.5 μg/mL of purified human Aβ peptide (H-1368, Bachem) in PBS and then incubated for 1 h under agitation at room temperature. .. After incubation, the plates were washed three times with PBS-Tween 0.05% (524653, Calbiochem) and blocked for 1 h at room temperature with 0.1% BSA solution (A7030, Sigma).

    Incubation:

    Article Title: Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model
    Article Snippet: Plasma Antibody QuantificationFor plasma concentration determination, standard 96-well sector plates (L15XA-1, Meso Scale Discovery) were coated with 0.5 μg/mL of purified extracellular domain of mTfR (50741-M07H, Sinobiological) or 0.5 μg/mL of purified human Aβ peptide (H-1368, Bachem) in PBS and then incubated for 1 h under agitation at room temperature. .. Plasma Antibody QuantificationFor plasma concentration determination, standard 96-well sector plates (L15XA-1, Meso Scale Discovery) were coated with 0.5 μg/mL of purified extracellular domain of mTfR (50741-M07H, Sinobiological) or 0.5 μg/mL of purified human Aβ peptide (H-1368, Bachem) in PBS and then incubated for 1 h under agitation at room temperature. .. After incubation, the plates were washed three times with PBS-Tween 0.05% (524653, Calbiochem) and blocked for 1 h at room temperature with 0.1% BSA solution (A7030, Sigma).

    Isolation:

    Article Title: Identification and in vivo characterization of a brain-penetrating nanobody
    Article Snippet: The library was transformed into electro-competent E.coli TG1 cells, which resulted in 108 independent transformants, of which 85% contained the vector with a right insert size. .. Isolation of anti-mTfR nanobodies To select anti-mTfR nanobodies, two rounds of in solution selections were performed with 100 and 50 nM biotinylated mTfR (50741-M07H-100, Sino Biological), respectively. ..

    Binding Assay:

    Article Title: Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles.
    Article Snippet: Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. .. Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. .. Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver.

    Recombinant:

    Article Title: Development of antibody-siRNA conjugate targeted to cardiac and skeletal muscles.
    Article Snippet: Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. .. Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. .. Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver.

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  • 93
    Sino Biological dcr 2
    Cross‐blocking studies with  DCR ‐2 and  UP ‐D2. Cells were incubated at the saturation point of a primary antibody or an equal concentration of an isotype control and then stained with  UP ‐D2  PE  at 80 ng·mL −1 . The  MFI  ratio is the comparison between primary antibody and isotype control groups. (A) The change in  UP ‐D2  PE  binding to  CD 300f C CHO  transfectants with different primary antibody staining. (B) Difference in  UP ‐D2 binding to  CD 300f SI 4 ‐ and  CD 300f C ‐transfected  CHO  cells, which were incubated with a saturating amount of  DCR ‐2 or isotype control prior to  UP ‐D2  PE . (C) Difference in  UP ‐D2 binding to  AML  cell lines, which were incubated with a saturating amount of  DCR ‐2 or isotype control prior to  UP ‐D2  PE . Error bars represent  SEM . Panel B was analyzed using a t‐test. Panel C was analyzed using a one‐way  ANOVA  with multiple comparisons between groups. *** P
    Dcr 2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dcr 2/product/Sino Biological
    Average 93 stars, based on 1 article reviews
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    94
    Sino Biological soluble cd38
    <t>CD38</t> and GDF15 expression and circulating levels are increased in AAA patients. ( A ) Human abdominal aortic mRNA levels of CD38 measured by quantitative real-time PCR and normalized to β-actin in healthy donors ( n = 15) and patients (AAA) ( n = 80). ( B ) CD38 plasma levels in AAA ( n = 94) vs. healthy donors ( n = 46); ( C ) plasma levels of GDF15 in AAA ( n = 94) vs. healthy donors ( n = 46). ( D ) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 ( n = 10; scale bars: 100 µm). ( E ) Representative images of immunostaining assays performed in abdominal aorta sections targeting GDF15 ( n = 0; scale bars: 100 µm). AAA (-) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells.
    Soluble Cd38, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/soluble cd38/product/Sino Biological
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    90
    Sino Biological recombinant cd146 elisa recombinant human cd146
    Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to <t>CD146.</t> A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see S5 Fig ). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.
    Recombinant Cd146 Elisa Recombinant Human Cd146, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant cd146 elisa recombinant human cd146/product/Sino Biological
    Average 90 stars, based on 1 article reviews
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    recombinant cd146 elisa recombinant human cd146 - by Bioz Stars, 2021-07
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    85
    Sino Biological human vwf elisa kit
    DIDS inhibits stimulus induced <t>vWF</t> release from normoxic HUVECs. The Ca 2+ ionophore A23187 induces vWF extrusion from HUVECs in a non-pathological model of vesicular release. DIDS abolishes stimulus-evoked vWF release. ( A ) Confocal Z-stack projection fluorescent images of vWF localization (red) in HUVECs treated as indicated. Arrows indicate vWF released extracellularly. ( B ) Summary of supernatant vWF expression measured by <t>ELISA.</t> Data are mean ±SEM from 3 separate 15-min experiments. Asterisks (*) indicate significant difference from normoxic controls; black bars indicate significance between connected treatments ( p
    Human Vwf Elisa Kit, supplied by Sino Biological, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    human vwf elisa kit - by Bioz Stars, 2021-07
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    Cross‐blocking studies with  DCR ‐2 and  UP ‐D2. Cells were incubated at the saturation point of a primary antibody or an equal concentration of an isotype control and then stained with  UP ‐D2  PE  at 80 ng·mL −1 . The  MFI  ratio is the comparison between primary antibody and isotype control groups. (A) The change in  UP ‐D2  PE  binding to  CD 300f C CHO  transfectants with different primary antibody staining. (B) Difference in  UP ‐D2 binding to  CD 300f SI 4 ‐ and  CD 300f C ‐transfected  CHO  cells, which were incubated with a saturating amount of  DCR ‐2 or isotype control prior to  UP ‐D2  PE . (C) Difference in  UP ‐D2 binding to  AML  cell lines, which were incubated with a saturating amount of  DCR ‐2 or isotype control prior to  UP ‐D2  PE . Error bars represent  SEM . Panel B was analyzed using a t‐test. Panel C was analyzed using a one‐way  ANOVA  with multiple comparisons between groups. *** P

    Journal: Molecular Oncology

    Article Title: CD300f epitopes are specific targets for acute myeloid leukemia with monocytic differentiation

    doi: 10.1002/1878-0261.12549

    Figure Lengend Snippet: Cross‐blocking studies with DCR ‐2 and UP ‐D2. Cells were incubated at the saturation point of a primary antibody or an equal concentration of an isotype control and then stained with UP ‐D2 PE at 80 ng·mL −1 . The MFI ratio is the comparison between primary antibody and isotype control groups. (A) The change in UP ‐D2 PE binding to CD 300f C CHO transfectants with different primary antibody staining. (B) Difference in UP ‐D2 binding to CD 300f SI 4 ‐ and CD 300f C ‐transfected CHO cells, which were incubated with a saturating amount of DCR ‐2 or isotype control prior to UP ‐D2 PE . (C) Difference in UP ‐D2 binding to AML cell lines, which were incubated with a saturating amount of DCR ‐2 or isotype control prior to UP ‐D2 PE . Error bars represent SEM . Panel B was analyzed using a t‐test. Panel C was analyzed using a one‐way ANOVA with multiple comparisons between groups. *** P

    Article Snippet: An in‐house mAb, DCR‐2 (IgG1,κ), was generated from a mouse immunized with CD300f Chinese hamster ovary (CHO) transfectants and boosted with recombinant human CD300f‐Fc protein (Sino Biologicals, Beijing, China).

    Techniques: Blocking Assay, Incubation, Concentration Assay, Staining, Binding Assay, Transfection

    Binding of  DCR ‐2 to  CD 300f +  cells enhances the binding of  UP ‐D2 to monocytes, monocytic  AML , but not  CD 34 + HSPC  or nonmonocytic  AML .  CB  or primary frozen  AML  cells were incubated with  PBS , the saturation point of  DCR ‐2 (10 μg·mL −1 ), or an equal concentration of an isotype control. Following primary incubation, samples were stained with  UP ‐D2  PE  at 80 ng·mL −1 . Data for monocytes, lymphocytes, and  CD 34 + HSPC  were obtained from  CB . (A) Difference in  UP ‐D2  PE  binding across cell types when saturated with  DCR ‐2 or isotype control, compared to  PBS . (B) Differences in  UP ‐D2  PE  binding across  CD 34 +  cells between  CB , monocytic  AML , and nonmonocytic  AML . Error bars represent  SEM . Statistical analysis was performed with one‐way  ANOVA  with multiple comparisons between groups. * P

    Journal: Molecular Oncology

    Article Title: CD300f epitopes are specific targets for acute myeloid leukemia with monocytic differentiation

    doi: 10.1002/1878-0261.12549

    Figure Lengend Snippet: Binding of DCR ‐2 to CD 300f + cells enhances the binding of UP ‐D2 to monocytes, monocytic AML , but not CD 34 + HSPC or nonmonocytic AML . CB or primary frozen AML cells were incubated with PBS , the saturation point of DCR ‐2 (10 μg·mL −1 ), or an equal concentration of an isotype control. Following primary incubation, samples were stained with UP ‐D2 PE at 80 ng·mL −1 . Data for monocytes, lymphocytes, and CD 34 + HSPC were obtained from CB . (A) Difference in UP ‐D2 PE binding across cell types when saturated with DCR ‐2 or isotype control, compared to PBS . (B) Differences in UP ‐D2 PE binding across CD 34 + cells between CB , monocytic AML , and nonmonocytic AML . Error bars represent SEM . Statistical analysis was performed with one‐way ANOVA with multiple comparisons between groups. * P

    Article Snippet: An in‐house mAb, DCR‐2 (IgG1,κ), was generated from a mouse immunized with CD300f Chinese hamster ovary (CHO) transfectants and boosted with recombinant human CD300f‐Fc protein (Sino Biologicals, Beijing, China).

    Techniques: Binding Assay, Incubation, Concentration Assay, Staining

    CD38 and GDF15 expression and circulating levels are increased in AAA patients. ( A ) Human abdominal aortic mRNA levels of CD38 measured by quantitative real-time PCR and normalized to β-actin in healthy donors ( n = 15) and patients (AAA) ( n = 80). ( B ) CD38 plasma levels in AAA ( n = 94) vs. healthy donors ( n = 46); ( C ) plasma levels of GDF15 in AAA ( n = 94) vs. healthy donors ( n = 46). ( D ) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 ( n = 10; scale bars: 100 µm). ( E ) Representative images of immunostaining assays performed in abdominal aorta sections targeting GDF15 ( n = 0; scale bars: 100 µm). AAA (-) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells.

    Journal: Antioxidants

    Article Title: Oxidative Stress and Inflammatory Markers in Abdominal Aortic Aneurysm

    doi: 10.3390/antiox10040602

    Figure Lengend Snippet: CD38 and GDF15 expression and circulating levels are increased in AAA patients. ( A ) Human abdominal aortic mRNA levels of CD38 measured by quantitative real-time PCR and normalized to β-actin in healthy donors ( n = 15) and patients (AAA) ( n = 80). ( B ) CD38 plasma levels in AAA ( n = 94) vs. healthy donors ( n = 46); ( C ) plasma levels of GDF15 in AAA ( n = 94) vs. healthy donors ( n = 46). ( D ) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 ( n = 10; scale bars: 100 µm). ( E ) Representative images of immunostaining assays performed in abdominal aorta sections targeting GDF15 ( n = 0; scale bars: 100 µm). AAA (-) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells.

    Article Snippet: ELISA KitsThe circulating levels of soluble IgM (ab214568, Abcam, UK), IgG (ab195215), soluble CD38 (KIT10818-1, Sino Biological Inc., Wayne, PA, USA), soluble CD36 (ABE-196-02, Nordic BioSite, Täby, Sweden), S100A4 (CSB-EL020632HU, Cusabio Biotech Co, LTD, Beijing, China) and GDF15 (Quantikine ELISA Human GDF15, DGD150; R & D Systems, Minneapolis, MN, USA), in plasma from patients were measured using commercially available ELISA kits in accordance with the manufacturer’s instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Negative Control, Immunohistochemistry, Staining

    IgG, CD38 and GDF15 circulating levels positively correlate with the AAA diameter whereas only CD38 correlates with PWS. ( A ) Graphs showing the correlation analysis between IgG plasma levels and AAA diameter ( n = 90) and ( B , C ) the correlation analysis between CD38 plasma levels and AAA diameter ( n = 90) or PWS values in AAA patients ( n = 58). ( D ) Graph showing the correlation analysis between GDF15 plasma levels and AAA diameter ( n = 90). The r and p -values are obtained by performing the Spearman or the Pearson correlation coefficient test. Results are expressed as mean ± SEM.

    Journal: Antioxidants

    Article Title: Oxidative Stress and Inflammatory Markers in Abdominal Aortic Aneurysm

    doi: 10.3390/antiox10040602

    Figure Lengend Snippet: IgG, CD38 and GDF15 circulating levels positively correlate with the AAA diameter whereas only CD38 correlates with PWS. ( A ) Graphs showing the correlation analysis between IgG plasma levels and AAA diameter ( n = 90) and ( B , C ) the correlation analysis between CD38 plasma levels and AAA diameter ( n = 90) or PWS values in AAA patients ( n = 58). ( D ) Graph showing the correlation analysis between GDF15 plasma levels and AAA diameter ( n = 90). The r and p -values are obtained by performing the Spearman or the Pearson correlation coefficient test. Results are expressed as mean ± SEM.

    Article Snippet: ELISA KitsThe circulating levels of soluble IgM (ab214568, Abcam, UK), IgG (ab195215), soluble CD38 (KIT10818-1, Sino Biological Inc., Wayne, PA, USA), soluble CD36 (ABE-196-02, Nordic BioSite, Täby, Sweden), S100A4 (CSB-EL020632HU, Cusabio Biotech Co, LTD, Beijing, China) and GDF15 (Quantikine ELISA Human GDF15, DGD150; R & D Systems, Minneapolis, MN, USA), in plasma from patients were measured using commercially available ELISA kits in accordance with the manufacturer’s instructions.

    Techniques:

    Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to CD146. A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see S5 Fig ). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.

    Journal: PLoS ONE

    Article Title: Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

    doi: 10.1371/journal.pone.0127169

    Figure Lengend Snippet: Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to CD146. A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see S5 Fig ). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.

    Article Snippet: Recombinant CD146 ELISA Recombinant human CD146 comprising the extracellular Met1 -Gly559 portion of the protein precursor (NP_006491.2) fused to a C-terminal poly-Histidine tag was purchased commercially (Sino Biological Inc., Nr.

    Techniques: Binding Assay, Western Blot, Clone Assay, Plasmid Preparation, Construct, Transfection, Cell Culture, SDS Page, Labeling, Produced, Immunoprecipitation, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Expressing, Purification, Standard Deviation

    LC-MS/MS identification of CD146 binding to scFv B6-11, and antigen confirmation by immunoprecipitation and ELISA. A: Alignment of LC-MS/MS identified peptides ( S4 Fig ) with the sequence of MCAM/CD146MUC18. The eluates from scFv B6-11 immunoprecipitation were subjected to trypsin digestion (see Materials and Methods section) and subsequently analyzed by LC-MS/MS. B: Immunoprecipitation of CD146 by soluble scFv B6-11 from BEC lysates. Immune complexes were tested by Western blot in reducing conditions using commercial anti-CD146 antibody. Lane 1: input BEC lysate, lanes 2–5: immunoprecipitates with scFv B6-11, lanes 2 and 4: under addition of PNGase F. Treatment of BEC lysates with PNGase prior or after addition of scFv B6-11 had no influence on co-immunoprecipitation capacity of scFv B6-11, showing that scFv B6-11 binding to CD146 is glycosylation-independent. C: scFv B6-11 binds to immobilized extracellular domain of recombinant human CD146 in ELISA. An irrelevant antigen (BSA), a non-binding scFv and uncoated wells served as controls. scFv binding was detected with peroxidase-conjugated anti-His tag antibody ( S1 Table ), and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. D: CD146 expression of different cell lines as shown by immunoprobing with anti-CD146 antibody. CD146 is expressed in BECs, in A375, CRL1676, HTB71 melanoma cells, but not in primary LECs and HEK293 cells. The same blot was probed with anti-tubulin antibody for control of equal protein loads. E: scFv B6-11 stains cell lines expressing CD146 with similar intensity as commercial anti-CD146 antibody in ELISA. Negative controls were a non-binding scFv and 2 nd antibody only. F: Similar to commercial anti-CD146 antibody, scFv B6-11 stains BECs (upper lane, red) but not LECs (lower lane, green) in immunofluorescence. Size bars: 50μm. G: scFv B6-11 stains blood, but not lymphatic vessels in human skin. Double immunofluorescence staining of skin sample with Cy3-labeled scFv-B6-11 or anti-CD146 (red) and anti-PDPN (green) antibodies. Blood (BV) and lymphatic (LV) vessels are indicated by lines. Nuclei were counterstained with DAPI. Size bars: 50μm.

    Journal: PLoS ONE

    Article Title: Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

    doi: 10.1371/journal.pone.0127169

    Figure Lengend Snippet: LC-MS/MS identification of CD146 binding to scFv B6-11, and antigen confirmation by immunoprecipitation and ELISA. A: Alignment of LC-MS/MS identified peptides ( S4 Fig ) with the sequence of MCAM/CD146MUC18. The eluates from scFv B6-11 immunoprecipitation were subjected to trypsin digestion (see Materials and Methods section) and subsequently analyzed by LC-MS/MS. B: Immunoprecipitation of CD146 by soluble scFv B6-11 from BEC lysates. Immune complexes were tested by Western blot in reducing conditions using commercial anti-CD146 antibody. Lane 1: input BEC lysate, lanes 2–5: immunoprecipitates with scFv B6-11, lanes 2 and 4: under addition of PNGase F. Treatment of BEC lysates with PNGase prior or after addition of scFv B6-11 had no influence on co-immunoprecipitation capacity of scFv B6-11, showing that scFv B6-11 binding to CD146 is glycosylation-independent. C: scFv B6-11 binds to immobilized extracellular domain of recombinant human CD146 in ELISA. An irrelevant antigen (BSA), a non-binding scFv and uncoated wells served as controls. scFv binding was detected with peroxidase-conjugated anti-His tag antibody ( S1 Table ), and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. D: CD146 expression of different cell lines as shown by immunoprobing with anti-CD146 antibody. CD146 is expressed in BECs, in A375, CRL1676, HTB71 melanoma cells, but not in primary LECs and HEK293 cells. The same blot was probed with anti-tubulin antibody for control of equal protein loads. E: scFv B6-11 stains cell lines expressing CD146 with similar intensity as commercial anti-CD146 antibody in ELISA. Negative controls were a non-binding scFv and 2 nd antibody only. F: Similar to commercial anti-CD146 antibody, scFv B6-11 stains BECs (upper lane, red) but not LECs (lower lane, green) in immunofluorescence. Size bars: 50μm. G: scFv B6-11 stains blood, but not lymphatic vessels in human skin. Double immunofluorescence staining of skin sample with Cy3-labeled scFv-B6-11 or anti-CD146 (red) and anti-PDPN (green) antibodies. Blood (BV) and lymphatic (LV) vessels are indicated by lines. Nuclei were counterstained with DAPI. Size bars: 50μm.

    Article Snippet: Recombinant CD146 ELISA Recombinant human CD146 comprising the extracellular Met1 -Gly559 portion of the protein precursor (NP_006491.2) fused to a C-terminal poly-Histidine tag was purchased commercially (Sino Biological Inc., Nr.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Binding Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Sequencing, Western Blot, Recombinant, Standard Deviation, Expressing, Immunofluorescence, Double Immunofluorescence Staining, Labeling

    DIDS inhibits stimulus induced vWF release from normoxic HUVECs. The Ca 2+ ionophore A23187 induces vWF extrusion from HUVECs in a non-pathological model of vesicular release. DIDS abolishes stimulus-evoked vWF release. ( A ) Confocal Z-stack projection fluorescent images of vWF localization (red) in HUVECs treated as indicated. Arrows indicate vWF released extracellularly. ( B ) Summary of supernatant vWF expression measured by ELISA. Data are mean ±SEM from 3 separate 15-min experiments. Asterisks (*) indicate significant difference from normoxic controls; black bars indicate significance between connected treatments ( p

    Journal: PLoS ONE

    Article Title: DIDS Prevents Ischemic Membrane Degradation in Cultured Hippocampal Neurons by Inhibiting Matrix Metalloproteinase Release

    doi: 10.1371/journal.pone.0043995

    Figure Lengend Snippet: DIDS inhibits stimulus induced vWF release from normoxic HUVECs. The Ca 2+ ionophore A23187 induces vWF extrusion from HUVECs in a non-pathological model of vesicular release. DIDS abolishes stimulus-evoked vWF release. ( A ) Confocal Z-stack projection fluorescent images of vWF localization (red) in HUVECs treated as indicated. Arrows indicate vWF released extracellularly. ( B ) Summary of supernatant vWF expression measured by ELISA. Data are mean ±SEM from 3 separate 15-min experiments. Asterisks (*) indicate significant difference from normoxic controls; black bars indicate significance between connected treatments ( p

    Article Snippet: ELISA HUVEC vWF release was quantified using a human vWF ELISA kit according to the manufacturer's protocol (Sino Biological Inc., Beijing, CH), using mouse anti-vWF monoclonal antibody and biotinylated rabbit anti-vWF polyclonal antibody as the capture and detection antibodies, respectively.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay