biotinylated protein a Search Results


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  • 94
    Vector Laboratories biotinylated protein a
    Targeted disruption of C1galt1 enhances endocytosis of <t>biotinylated</t> cell surface protein in corneal keratinocytes. (A) Cell surface protein was labeled with biotin at 4°C and then allowed to internalize for 15 or 25 min at 37°C. Crude postnuclear supernatants were ultracentrifuged using 5–25% Optiprep gradients and analyzed by agarose gel electrophoresis. Fraction 1 contains the lightest membranes; fraction 16 contains the densest membranes. The position of the plasma membrane and endocytic vesicles in the gradient was determined by western blot using antibodies to MUC16 and human TfR, respectively. Biotinylated protein was detected using streptavidin-peroxidase as described in Materials and Methods . (B) Quantitative evaluation of biotin accumulation in Optiprep density gradient. The graph shows the ratio of biotinylated protein in C1galt1 shRNA and scramble shRNA cells as determined by densitometry. Band intensity in each fraction was normalized to percent total biotin loaded in each gradient. Data for each condition are reported as the mean of three independent experiments.
    Biotinylated Protein A, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher biotinylated
    Noggin does not affect association of LPP with BMP-RII in IVD cells. Cells were incubated with a <t>biotinylated</t> peptide LPPb in the in the absence or presence of noggin (600 ng/ml) and compared with control cells. After a 48-h incubation, cells were washed,
    Biotinylated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1783 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher biotinylated protein a
    Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using <t>biotinylated</t> <t>Protein</t> A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.
    Biotinylated Protein A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore biotinylated protein a
    Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using <t>biotinylated</t> <t>Protein</t> A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.
    Biotinylated Protein A, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher recombinant biotinylated protein a protein g
    AP-1 adaptors are necessary for endosomal vesicle formation in vitro. (A) Bovine brain cytosol was immunodepleted of AP-1 by using anti-γ-adaptin antibody and protein <t>A/G-Sepharose</t> beads. Total cytosol (T) and corresponding aliquots of the depleted cytosol (unbound fraction, U) and the bound material (B) were analyzed by immunoblotting with anti-γ-adaptin antibody. Depletion efficiency was > 80%. The positions of molecular weight markers are indicated (in kilodaltons). (B) Biotinylated permeabilized cells were incubated in the presence of ATP, GTP, and ATP-regenerating system without cytosol (no cyt), with untreated cytosol (cyt; 1.2 mg/ml), with AP-1– depleted cytosol (cyt–AP-1), with AP-1– depleted cytosol that had been supplemented with 12 μg/ml purified AP-1 (cyt–AP-1+AP-1), or with mock-depleted cytosol. Immunoblot analysis of biotinylated H1 in the supernatant after cell pelleting is shown for a representative experiment. Quantitation of three independent experiments (average with SD) is presented below.
    Recombinant Biotinylated Protein A Protein G, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    r&d systems biotinylated protein a
    AP-1 adaptors are necessary for endosomal vesicle formation in vitro. (A) Bovine brain cytosol was immunodepleted of AP-1 by using anti-γ-adaptin antibody and protein <t>A/G-Sepharose</t> beads. Total cytosol (T) and corresponding aliquots of the depleted cytosol (unbound fraction, U) and the bound material (B) were analyzed by immunoblotting with anti-γ-adaptin antibody. Depletion efficiency was > 80%. The positions of molecular weight markers are indicated (in kilodaltons). (B) Biotinylated permeabilized cells were incubated in the presence of ATP, GTP, and ATP-regenerating system without cytosol (no cyt), with untreated cytosol (cyt; 1.2 mg/ml), with AP-1– depleted cytosol (cyt–AP-1), with AP-1– depleted cytosol that had been supplemented with 12 μg/ml purified AP-1 (cyt–AP-1+AP-1), or with mock-depleted cytosol. Immunoblot analysis of biotinylated H1 in the supernatant after cell pelleting is shown for a representative experiment. Quantitation of three independent experiments (average with SD) is presented below.
    Biotinylated Protein A, supplied by r&d systems, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Abcam biotinylated anti protein a antibody
    AP-1 adaptors are necessary for endosomal vesicle formation in vitro. (A) Bovine brain cytosol was immunodepleted of AP-1 by using anti-γ-adaptin antibody and protein <t>A/G-Sepharose</t> beads. Total cytosol (T) and corresponding aliquots of the depleted cytosol (unbound fraction, U) and the bound material (B) were analyzed by immunoblotting with anti-γ-adaptin antibody. Depletion efficiency was > 80%. The positions of molecular weight markers are indicated (in kilodaltons). (B) Biotinylated permeabilized cells were incubated in the presence of ATP, GTP, and ATP-regenerating system without cytosol (no cyt), with untreated cytosol (cyt; 1.2 mg/ml), with AP-1– depleted cytosol (cyt–AP-1), with AP-1– depleted cytosol that had been supplemented with 12 μg/ml purified AP-1 (cyt–AP-1+AP-1), or with mock-depleted cytosol. Immunoblot analysis of biotinylated H1 in the supernatant after cell pelleting is shown for a representative experiment. Quantitation of three independent experiments (average with SD) is presented below.
    Biotinylated Anti Protein A Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore biotinylated native protein a p2165
    Representative sequences of the 15 identified groups from the NGS pool were screened for their individual binding abilities to Protein A. Comparative SPR-based interaction analyses were performed with the Biacore X100 instrument. <t>Biotinylated</t> aptamers were immobilized via the 5′-end ( A ) or 3′-end ( B ) on the streptavidin-modified sensor surface and 1000 nM <t>Protein</t> A was injected for binding. The sensor responses from the end of the binding phases (after 300 s) are shown. In addition, cross-specificities to other proteins were analyzed. Asterisks indicate if certain interactions have not been investigated.
    Biotinylated Native Protein A P2165, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Sino Biological biotinylated recombinant
    The impact of the recombinant <t>biotinylated</t> CD38-DARA complex on the endogenous M-protein quantification in six patients with a range of endogenous M-protein concentration (0.3–2.9 g/dL) was assessed based on the correlation of the quantitation pre- and post-CD38 treatment (R squared = 0.9984) .
    Biotinylated Recombinant, 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
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    94
    BioVision biotinylated protein a g
    The impact of the recombinant <t>biotinylated</t> CD38-DARA complex on the endogenous M-protein quantification in six patients with a range of endogenous M-protein concentration (0.3–2.9 g/dL) was assessed based on the correlation of the quantitation pre- and post-CD38 treatment (R squared = 0.9984) .
    Biotinylated Protein A G, supplied by BioVision, used in various techniques. Bioz Stars score: 94/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 29 article reviews
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    99
    Thermo Fisher protein a agarose
    The impact of the recombinant <t>biotinylated</t> CD38-DARA complex on the endogenous M-protein quantification in six patients with a range of endogenous M-protein concentration (0.3–2.9 g/dL) was assessed based on the correlation of the quantitation pre- and post-CD38 treatment (R squared = 0.9984) .
    Protein A Agarose, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3020 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore staphylococcal protein a
    The impact of the recombinant <t>biotinylated</t> CD38-DARA complex on the endogenous M-protein quantification in six patients with a range of endogenous M-protein concentration (0.3–2.9 g/dL) was assessed based on the correlation of the quantitation pre- and post-CD38 treatment (R squared = 0.9984) .
    Staphylococcal Protein A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore biotinylated spa
    514G3 antibody binds to WT SpA with specificity and picomolar affinity. A. Antigen binding profiles of 514G3 captured on an active flow cell on a Biacore 3000. The colored lines on the sensogram represent the recorded binding response signals at different antigen concentrations from 2-0nM, and the overlaid black lines represent the fitted curves. Binding of SpA to 514G3 was monitored in real time to obtain on (ka) and off (kd) rates. The equilibrium constant (K D ) was calculated from the observed Ka and Kd and found to be 46.7pM. B. A graph showing the relative binding of 514G3 to SpA, MabSelect and MabSelect SuRe ligands. The three different SpA domain variants were coated on the wells of an ELISA plate, and then probed with <t>biotinylated</t> 514G3 or VH3/IgG3-k isotype control at 4μg/ml, and detected with streptavidin-HRP. The graph shows OD at 450nm on the y-axis and the different SpA domain variants on the X-axis. The error bars show the standard deviation from three replicates.
    Biotinylated Spa, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Targeted disruption of C1galt1 enhances endocytosis of biotinylated cell surface protein in corneal keratinocytes. (A) Cell surface protein was labeled with biotin at 4°C and then allowed to internalize for 15 or 25 min at 37°C. Crude postnuclear supernatants were ultracentrifuged using 5–25% Optiprep gradients and analyzed by agarose gel electrophoresis. Fraction 1 contains the lightest membranes; fraction 16 contains the densest membranes. The position of the plasma membrane and endocytic vesicles in the gradient was determined by western blot using antibodies to MUC16 and human TfR, respectively. Biotinylated protein was detected using streptavidin-peroxidase as described in Materials and Methods . (B) Quantitative evaluation of biotin accumulation in Optiprep density gradient. The graph shows the ratio of biotinylated protein in C1galt1 shRNA and scramble shRNA cells as determined by densitometry. Band intensity in each fraction was normalized to percent total biotin loaded in each gradient. Data for each condition are reported as the mean of three independent experiments.

    Journal: PLoS ONE

    Article Title: Targeted Disruption of Core 1 ?1,3-galactosyltransferase (C1galt1) Induces Apical Endocytic Trafficking in Human Corneal Keratinocytes

    doi: 10.1371/journal.pone.0036628

    Figure Lengend Snippet: Targeted disruption of C1galt1 enhances endocytosis of biotinylated cell surface protein in corneal keratinocytes. (A) Cell surface protein was labeled with biotin at 4°C and then allowed to internalize for 15 or 25 min at 37°C. Crude postnuclear supernatants were ultracentrifuged using 5–25% Optiprep gradients and analyzed by agarose gel electrophoresis. Fraction 1 contains the lightest membranes; fraction 16 contains the densest membranes. The position of the plasma membrane and endocytic vesicles in the gradient was determined by western blot using antibodies to MUC16 and human TfR, respectively. Biotinylated protein was detected using streptavidin-peroxidase as described in Materials and Methods . (B) Quantitative evaluation of biotin accumulation in Optiprep density gradient. The graph shows the ratio of biotinylated protein in C1galt1 shRNA and scramble shRNA cells as determined by densitometry. Band intensity in each fraction was normalized to percent total biotin loaded in each gradient. Data for each condition are reported as the mean of three independent experiments.

    Article Snippet: Biotinylated protein was detected on the membranes after blocking with Tris-buffered saline Tween (TBS-T) using a biotin-streptavidin-peroxidase kit (Vectastain® ABC Kit; Vector Laboratories, Burlingame, CA).

    Techniques: Labeling, Agarose Gel Electrophoresis, Western Blot, shRNA

    Association of the FcRγ chain with the NKR-P1 molecule in IL-2–expanded NK cells. NK cells from normal mice expanded for 1 wk in the presence of IL-2 and were surface-biotinylated. The cell lysate of them was immunoprecipitated with anti-NK1.1 mAb ((Fab)′ 2 fragment) and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected as Fig. 1 A. FcRγ homodimer ( γ - γ ) was indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: Association with FcR? Is Essential for Activation Signal through NKR-P1 (CD161) in Natural Killer (NK) Cells and NK1.1+ T Cells

    doi:

    Figure Lengend Snippet: Association of the FcRγ chain with the NKR-P1 molecule in IL-2–expanded NK cells. NK cells from normal mice expanded for 1 wk in the presence of IL-2 and were surface-biotinylated. The cell lysate of them was immunoprecipitated with anti-NK1.1 mAb ((Fab)′ 2 fragment) and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected as Fig. 1 A. FcRγ homodimer ( γ - γ ) was indicated.

    Article Snippet: The biotinylated proteins were detected using streptavidin-peroxidase (VECSTAIN Elite ABC kit; Vector Laboratories Incorporated, Burlingame, CA), the ECL system ( Amersham International , Buckinghamshire, England), and autoradiography.

    Techniques: Mouse Assay, Immunoprecipitation, SDS Page

    Association of the FcRγ chain with the NKR-P1 molecule on the cell surface of CTLL-2 cell line. ( A ) CTLL-2 cells were surface-biotinylated and the cell lysate was immunoprecipitated with anti-NK1.1 mAb or anti-CD3ε mAb and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected by an ECL. ( B ) FcRγ chain was detected by blotting the membrane with anti-FcRγ Ab after immunoprecipitation with anti-NK1.1 or anti-CD3ε mAbs. FcRγ homodimer ( γ - γ ) and CD3ζ-FcRγ heterodimers ( ζ - γ ) were indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: Association with FcR? Is Essential for Activation Signal through NKR-P1 (CD161) in Natural Killer (NK) Cells and NK1.1+ T Cells

    doi:

    Figure Lengend Snippet: Association of the FcRγ chain with the NKR-P1 molecule on the cell surface of CTLL-2 cell line. ( A ) CTLL-2 cells were surface-biotinylated and the cell lysate was immunoprecipitated with anti-NK1.1 mAb or anti-CD3ε mAb and analyzed on two-dimensional nonreducing ( NR ) and reducing ( R ) SDS-PAGE. Biotinylated proteins were detected by an ECL. ( B ) FcRγ chain was detected by blotting the membrane with anti-FcRγ Ab after immunoprecipitation with anti-NK1.1 or anti-CD3ε mAbs. FcRγ homodimer ( γ - γ ) and CD3ζ-FcRγ heterodimers ( ζ - γ ) were indicated.

    Article Snippet: The biotinylated proteins were detected using streptavidin-peroxidase (VECSTAIN Elite ABC kit; Vector Laboratories Incorporated, Burlingame, CA), the ECL system ( Amersham International , Buckinghamshire, England), and autoradiography.

    Techniques: Immunoprecipitation, SDS Page

    Modification of the CD3γ subunit after in vitro activation and expansion. (A) 30 × 10 6 ex vivo γδ (γδ TCR Tg [Tg] CD3δ +/− mice) and stimulated γδ T cells (Tg CD3δ +/− and Tg CD3δ −/− mice) were surface biotinylated, lysed, and incubated with anti-CD3ɛ mAb (145–2C 11 ). Immunoprecipitated proteins were either treated with PNGase F or left untreated and resolved by reducing SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked. (B) Lysates from 30 × 10 6 ex vivo γδ (Tg CD3δ +/− mice) and stimulated γδ T cells (Tg CD3δ +/− and Tg CD3δ −/− mice) were immunoprecipitated with anti-TCR-ζ mAb (H146) and then treated with PNGase F or left untreated. Digested and undigested TCR proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum. (C) Lysates from 30 × 10 6 ex vivo αβ (B6 mice) and stimulated αβ T cells (B6 and CD3δ −/− mice) were immunoprecipitated with anti-CD3ɛ (145–2C 11 ) or anti-TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum. (D) Stimulated αβ and γδ T cells from CD3δ 2 / − mice were generated as described in Materials and Methods. Surface proteins on equivalent numbers of cells were labeled with biotin and TCR complexes were immunoprecipitated using the anti-CD3ɛ mAb (145–2C 11 ). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The exposure time for each blot is identical. The positions of the CD3ɛ and the modified forms of CD3γ are marked.

    Journal: The Journal of Experimental Medicine

    Article Title: Activation-induced Modification in the CD3 Complex of the ?? T Cell Receptor

    doi: 10.1084/jem.20021196

    Figure Lengend Snippet: Modification of the CD3γ subunit after in vitro activation and expansion. (A) 30 × 10 6 ex vivo γδ (γδ TCR Tg [Tg] CD3δ +/− mice) and stimulated γδ T cells (Tg CD3δ +/− and Tg CD3δ −/− mice) were surface biotinylated, lysed, and incubated with anti-CD3ɛ mAb (145–2C 11 ). Immunoprecipitated proteins were either treated with PNGase F or left untreated and resolved by reducing SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked. (B) Lysates from 30 × 10 6 ex vivo γδ (Tg CD3δ +/− mice) and stimulated γδ T cells (Tg CD3δ +/− and Tg CD3δ −/− mice) were immunoprecipitated with anti-TCR-ζ mAb (H146) and then treated with PNGase F or left untreated. Digested and undigested TCR proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum. (C) Lysates from 30 × 10 6 ex vivo αβ (B6 mice) and stimulated αβ T cells (B6 and CD3δ −/− mice) were immunoprecipitated with anti-CD3ɛ (145–2C 11 ) or anti-TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by reducing SDS-PAGE and immunoblotted with anti-CD3γ serum. (D) Stimulated αβ and γδ T cells from CD3δ 2 / − mice were generated as described in Materials and Methods. Surface proteins on equivalent numbers of cells were labeled with biotin and TCR complexes were immunoprecipitated using the anti-CD3ɛ mAb (145–2C 11 ). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The exposure time for each blot is identical. The positions of the CD3ɛ and the modified forms of CD3γ are marked.

    Article Snippet: Biotinylated surface proteins were detected using ABC-HRP (Vector Laboratories) and the ECL Western blot detection system (Amersham Pharmacia Biotech).

    Techniques: Modification, In Vitro, Activation Assay, Ex Vivo, Mouse Assay, Incubation, Immunoprecipitation, SDS Page, Generated, Labeling

    Effect of in vitro activation and expansion on the subunit composition of αβ- and γδ TCR complexes. Ex vivo αβ and γδ T cells were purified from the lymph nodes of B6 and γδ TCR Tg mice, respectively. Stimulated αβ and γδ T cells, from B6 and γδ TCR Tg mice, respectively, were generated as described in Materials and Methods. Surface proteins were labeled with biotin and αβ and γδ TCR complexes were immunoprecipitated using anti-TCR mAbs (H57–597 and UC7–13D5, respectively) or anti-TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked.

    Journal: The Journal of Experimental Medicine

    Article Title: Activation-induced Modification in the CD3 Complex of the ?? T Cell Receptor

    doi: 10.1084/jem.20021196

    Figure Lengend Snippet: Effect of in vitro activation and expansion on the subunit composition of αβ- and γδ TCR complexes. Ex vivo αβ and γδ T cells were purified from the lymph nodes of B6 and γδ TCR Tg mice, respectively. Stimulated αβ and γδ T cells, from B6 and γδ TCR Tg mice, respectively, were generated as described in Materials and Methods. Surface proteins were labeled with biotin and αβ and γδ TCR complexes were immunoprecipitated using anti-TCR mAbs (H57–597 and UC7–13D5, respectively) or anti-TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. ABC-HRP and chemiluminescence were used to visualize surface biotinylated proteins. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked.

    Article Snippet: Biotinylated surface proteins were detected using ABC-HRP (Vector Laboratories) and the ECL Western blot detection system (Amersham Pharmacia Biotech).

    Techniques: In Vitro, Activation Assay, Ex Vivo, Purification, Mouse Assay, Generated, Labeling, Immunoprecipitation, SDS Page

    Biochemical analysis of CD3 complexes expressed by stimulated γδ T cells. (A) Lysates from 20 × 10 6 ex vivo αβ and γδ T cells and stimulated γδ T cells were immunoprecipitated with anti–TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and probed with anti-CD3ɛ to assess the efficiency of immunoprecipitation. (B) Total cellular CD3δ protein levels in ex vivo and in stimulated αβ and γδ T cells. Extracts were made from 10 × 10 6 cells and 0.3 × 10 6 cell equivalents were then analyzed by immunoblotting with anti-CD3δ serum. (C) Lysates from 25 × 10 6 stimulated V γ 1-J γ 4-C γ 4 + γδ T cells and B6 thymocytes were immunoprecipitated with anti-TCR mAbs (UC7–13D5 and H57–597, respectively). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and probed with anti-CD3ɛ to assess the efficiency of immunoprecipitation. (D) 20 × 10 6 stimulated γδ T cells from nontransgenic CD3δ −/− mice were surface biotinylated, lysed, and incubated with anti-CD3ɛ mAb (145–2C 11 ). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. Biotinylated TCR subunits were detected with ABC-HRP and chemiluminescence. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked.

    Journal: The Journal of Experimental Medicine

    Article Title: Activation-induced Modification in the CD3 Complex of the ?? T Cell Receptor

    doi: 10.1084/jem.20021196

    Figure Lengend Snippet: Biochemical analysis of CD3 complexes expressed by stimulated γδ T cells. (A) Lysates from 20 × 10 6 ex vivo αβ and γδ T cells and stimulated γδ T cells were immunoprecipitated with anti–TCR-ζ mAb (H146). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and probed with anti-CD3ɛ to assess the efficiency of immunoprecipitation. (B) Total cellular CD3δ protein levels in ex vivo and in stimulated αβ and γδ T cells. Extracts were made from 10 × 10 6 cells and 0.3 × 10 6 cell equivalents were then analyzed by immunoblotting with anti-CD3δ serum. (C) Lysates from 25 × 10 6 stimulated V γ 1-J γ 4-C γ 4 + γδ T cells and B6 thymocytes were immunoprecipitated with anti-TCR mAbs (UC7–13D5 and H57–597, respectively). Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-CD3δ serum. The blot was subsequently stripped and probed with anti-CD3ɛ to assess the efficiency of immunoprecipitation. (D) 20 × 10 6 stimulated γδ T cells from nontransgenic CD3δ −/− mice were surface biotinylated, lysed, and incubated with anti-CD3ɛ mAb (145–2C 11 ). Immunoprecipitated proteins were resolved by nonreducing/reducing 2-D SDS-PAGE. Biotinylated TCR subunits were detected with ABC-HRP and chemiluminescence. The positions of the CD3 subunits and the unknown 26-kD subunit (?) are marked.

    Article Snippet: Biotinylated surface proteins were detected using ABC-HRP (Vector Laboratories) and the ECL Western blot detection system (Amersham Pharmacia Biotech).

    Techniques: Ex Vivo, Immunoprecipitation, SDS Page, Mouse Assay, Incubation

    Noggin does not affect association of LPP with BMP-RII in IVD cells. Cells were incubated with a biotinylated peptide LPPb in the in the absence or presence of noggin (600 ng/ml) and compared with control cells. After a 48-h incubation, cells were washed,

    Journal: The Journal of Biological Chemistry

    Article Title: Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells *

    doi: 10.1074/jbc.M113.451948

    Figure Lengend Snippet: Noggin does not affect association of LPP with BMP-RII in IVD cells. Cells were incubated with a biotinylated peptide LPPb in the in the absence or presence of noggin (600 ng/ml) and compared with control cells. After a 48-h incubation, cells were washed,

    Article Snippet: To capture biotinylated LPP-bound protein, the supernatant containing biotinylated peptide-protein conjugates was passed through a monomeric avidin-Sepharose column (Pierce).

    Techniques: Incubation

    LPP directly binds to BMP-RII in IVD cells. Cells were incubated with a biotinylated peptide LPPb or a biotinylated control peptide LPRb and compared with untreated cells ( Ctrl ). A , after a 48-h incubation, cells were labeled with Alexa Fluor 568-conjugated

    Journal: The Journal of Biological Chemistry

    Article Title: Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells *

    doi: 10.1074/jbc.M113.451948

    Figure Lengend Snippet: LPP directly binds to BMP-RII in IVD cells. Cells were incubated with a biotinylated peptide LPPb or a biotinylated control peptide LPRb and compared with untreated cells ( Ctrl ). A , after a 48-h incubation, cells were labeled with Alexa Fluor 568-conjugated

    Article Snippet: To capture biotinylated LPP-bound protein, the supernatant containing biotinylated peptide-protein conjugates was passed through a monomeric avidin-Sepharose column (Pierce).

    Techniques: Incubation, Labeling

    Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using biotinylated Protein A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.

    Journal: Protein Expression and Purification

    Article Title: Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli

    doi: 10.1016/j.pep.2015.07.008

    Figure Lengend Snippet: Immunoreactivity of recombinant henipavirus nucleocapsid protein and soluble glycoprotein towards different anti-sera from infected and non-infected organisms in a Luminex bead-based assay. Detection of antibodies to recombinant henipavirus N and sG proteins in sera from animals and humans infected naturally (NI) or laboratory infected (LI) with, either HeV, NiV or CedPV, and horses receiving Equivac® HeV vaccine (Vac). Non-infected humans and animals were designated normal. HeV N FL and HeV N CORE together HeV, NiV and CedPV sG were coupled to individual sets of Luminex beads. Binding of specific antibodies were detected using biotinylated Protein A and biotinylated Protein G and streptavidin–phycoerythrin. The results were recorded as the Mean Fluorescent Intensity (MFI) of 100 beads.

    Article Snippet: The bound antibodies were then detected using biotinylated Protein A (Pierce) and biotinylated Protein G (Pierce) and streptavidin–phycoerythrin (Qiagen).

    Techniques: Recombinant, Infection, Luminex, Bead-based Assay, Binding Assay

    AP-1 adaptors are necessary for endosomal vesicle formation in vitro. (A) Bovine brain cytosol was immunodepleted of AP-1 by using anti-γ-adaptin antibody and protein A/G-Sepharose beads. Total cytosol (T) and corresponding aliquots of the depleted cytosol (unbound fraction, U) and the bound material (B) were analyzed by immunoblotting with anti-γ-adaptin antibody. Depletion efficiency was > 80%. The positions of molecular weight markers are indicated (in kilodaltons). (B) Biotinylated permeabilized cells were incubated in the presence of ATP, GTP, and ATP-regenerating system without cytosol (no cyt), with untreated cytosol (cyt; 1.2 mg/ml), with AP-1– depleted cytosol (cyt–AP-1), with AP-1– depleted cytosol that had been supplemented with 12 μg/ml purified AP-1 (cyt–AP-1+AP-1), or with mock-depleted cytosol. Immunoblot analysis of biotinylated H1 in the supernatant after cell pelleting is shown for a representative experiment. Quantitation of three independent experiments (average with SD) is presented below.

    Journal: Molecular Biology of the Cell

    Article Title: In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

    doi: 10.1091/mbc.E04-04-0355

    Figure Lengend Snippet: AP-1 adaptors are necessary for endosomal vesicle formation in vitro. (A) Bovine brain cytosol was immunodepleted of AP-1 by using anti-γ-adaptin antibody and protein A/G-Sepharose beads. Total cytosol (T) and corresponding aliquots of the depleted cytosol (unbound fraction, U) and the bound material (B) were analyzed by immunoblotting with anti-γ-adaptin antibody. Depletion efficiency was > 80%. The positions of molecular weight markers are indicated (in kilodaltons). (B) Biotinylated permeabilized cells were incubated in the presence of ATP, GTP, and ATP-regenerating system without cytosol (no cyt), with untreated cytosol (cyt; 1.2 mg/ml), with AP-1– depleted cytosol (cyt–AP-1), with AP-1– depleted cytosol that had been supplemented with 12 μg/ml purified AP-1 (cyt–AP-1+AP-1), or with mock-depleted cytosol. Immunoblot analysis of biotinylated H1 in the supernatant after cell pelleting is shown for a representative experiment. Quantitation of three independent experiments (average with SD) is presented below.

    Article Snippet: Adenylyl imidodiphosphate (AMP-PNP), guanylyl imidodiphosphate (GMP-PNP), ATP, GTP, creatine kinase, and creatine phosphate were from Roche Diagnostics (Indianapolis, IN); sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin) and avidin-Sepharose were from Pierce Chemical (Rockford, IL); protein A-Sepharose was from Zymed Laboratories (South San Francisco, CA), protein G-Sepharose was from Gerbu (Gaiberg, Germany); and was from BIOMOL Research Laboratories (Plymouth Meeting, PA).

    Techniques: In Vitro, Molecular Weight, Incubation, Purification, Quantitation Assay

    Representative sequences of the 15 identified groups from the NGS pool were screened for their individual binding abilities to Protein A. Comparative SPR-based interaction analyses were performed with the Biacore X100 instrument. Biotinylated aptamers were immobilized via the 5′-end ( A ) or 3′-end ( B ) on the streptavidin-modified sensor surface and 1000 nM Protein A was injected for binding. The sensor responses from the end of the binding phases (after 300 s) are shown. In addition, cross-specificities to other proteins were analyzed. Asterisks indicate if certain interactions have not been investigated.

    Journal: International Journal of Molecular Sciences

    Article Title: Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

    doi: 10.3390/ijms19020642

    Figure Lengend Snippet: Representative sequences of the 15 identified groups from the NGS pool were screened for their individual binding abilities to Protein A. Comparative SPR-based interaction analyses were performed with the Biacore X100 instrument. Biotinylated aptamers were immobilized via the 5′-end ( A ) or 3′-end ( B ) on the streptavidin-modified sensor surface and 1000 nM Protein A was injected for binding. The sensor responses from the end of the binding phases (after 300 s) are shown. In addition, cross-specificities to other proteins were analyzed. Asterisks indicate if certain interactions have not been investigated.

    Article Snippet: Materials Native Protein A from S. aureus (P3838), biotinylated native Protein A (P2165), and recombinant Protein A (P7837, expressed in E. coli ), as well as human serum albumin (HSA, A9511) and bovine serum albumin (BSA, A3059) were purchased from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: Next-Generation Sequencing, Binding Assay, SPR Assay, Modification, Injection

    SPR-based interaction analyses with the Biacore X100 instrument applying aptamers as analyte. Biotinylated native Protein A was immobilized as ligand on the streptavidin-modified sensor surface. ( A ) Comparable binding analysis of unmodified candidate aptamers, 2000 nM each; ( B ) A concentration series of aptamer PA#2/8 was applied for binding. Black lines represent the fit to two state reaction model. The corresponding plot of steady-state binding data from the end of the association phases against analyte concentrations were used to calculate the steady-state affinity ( K D ).

    Journal: International Journal of Molecular Sciences

    Article Title: Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

    doi: 10.3390/ijms19020642

    Figure Lengend Snippet: SPR-based interaction analyses with the Biacore X100 instrument applying aptamers as analyte. Biotinylated native Protein A was immobilized as ligand on the streptavidin-modified sensor surface. ( A ) Comparable binding analysis of unmodified candidate aptamers, 2000 nM each; ( B ) A concentration series of aptamer PA#2/8 was applied for binding. Black lines represent the fit to two state reaction model. The corresponding plot of steady-state binding data from the end of the association phases against analyte concentrations were used to calculate the steady-state affinity ( K D ).

    Article Snippet: Materials Native Protein A from S. aureus (P3838), biotinylated native Protein A (P2165), and recombinant Protein A (P7837, expressed in E. coli ), as well as human serum albumin (HSA, A9511) and bovine serum albumin (BSA, A3059) were purchased from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: SPR Assay, Modification, Binding Assay, Concentration Assay

    SPR-based interaction analyses with the Biacore X100 instrument regarding the affinity of aptameric sequences from group 1 ( A – C ), 2 ( D , E ) and 6 ( F ) to Protein A. Biotinylated aptamers were immobilized via the 5′- or 3′-end on the streptavidin-modified sensor surface and a concentration series of recombinant or native Protein A was applied for binding. Black lines represent the fit to bivalent analyte binding model (group 1 and 6 aptamers) or to two state reaction model (group 2 aptamers). The corresponding plots of steady-state binding data from the end of the association phases against analyte concentrations were used to calculate the steady-state affinities ( K D ).

    Journal: International Journal of Molecular Sciences

    Article Title: Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

    doi: 10.3390/ijms19020642

    Figure Lengend Snippet: SPR-based interaction analyses with the Biacore X100 instrument regarding the affinity of aptameric sequences from group 1 ( A – C ), 2 ( D , E ) and 6 ( F ) to Protein A. Biotinylated aptamers were immobilized via the 5′- or 3′-end on the streptavidin-modified sensor surface and a concentration series of recombinant or native Protein A was applied for binding. Black lines represent the fit to bivalent analyte binding model (group 1 and 6 aptamers) or to two state reaction model (group 2 aptamers). The corresponding plots of steady-state binding data from the end of the association phases against analyte concentrations were used to calculate the steady-state affinities ( K D ).

    Article Snippet: Materials Native Protein A from S. aureus (P3838), biotinylated native Protein A (P2165), and recombinant Protein A (P7837, expressed in E. coli ), as well as human serum albumin (HSA, A9511) and bovine serum albumin (BSA, A3059) were purchased from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: SPR Assay, Modification, Concentration Assay, Recombinant, Binding Assay

    The impact of the recombinant biotinylated CD38-DARA complex on the endogenous M-protein quantification in six patients with a range of endogenous M-protein concentration (0.3–2.9 g/dL) was assessed based on the correlation of the quantitation pre- and post-CD38 treatment (R squared = 0.9984) .

    Journal: Diagnostics

    Article Title: Complete Depletion of Daratumumab Interference in Serum Samples from Plasma Cell Myeloma Patients Improves the Detection of Endogenous M-Proteins in a Preliminary Study

    doi: 10.3390/diagnostics10040219

    Figure Lengend Snippet: The impact of the recombinant biotinylated CD38-DARA complex on the endogenous M-protein quantification in six patients with a range of endogenous M-protein concentration (0.3–2.9 g/dL) was assessed based on the correlation of the quantitation pre- and post-CD38 treatment (R squared = 0.9984) .

    Article Snippet: The spiked sera aliquots were then supplemented with 0.125–0.5 g/L of biotinylated recombinant, human CD38 (Sino Biological, city, country, Catalog number: 10818-H08H-B) and incubated for ten minutes on an end-over-end tube rotator at room temperature.

    Techniques: Recombinant, Protein Concentration, Quantitation Assay

    Schematic illustration of steps of a novel immunoaffinity method to deplete residual daratumumab (DARA) using biotinylated recombinant full length CD38.

    Journal: Diagnostics

    Article Title: Complete Depletion of Daratumumab Interference in Serum Samples from Plasma Cell Myeloma Patients Improves the Detection of Endogenous M-Proteins in a Preliminary Study

    doi: 10.3390/diagnostics10040219

    Figure Lengend Snippet: Schematic illustration of steps of a novel immunoaffinity method to deplete residual daratumumab (DARA) using biotinylated recombinant full length CD38.

    Article Snippet: The spiked sera aliquots were then supplemented with 0.125–0.5 g/L of biotinylated recombinant, human CD38 (Sino Biological, city, country, Catalog number: 10818-H08H-B) and incubated for ten minutes on an end-over-end tube rotator at room temperature.

    Techniques: Recombinant

    Pooled patient sera with no endogenous monoclonal immunoglobulin protein (M-protein) spiked with DARA resulted in a clear IgG (G) kappa (K) M-protein (indicated by arrows) on serum immunofixation (sIFE) gel. Incubation of DARA-spiked serum with 0.125–0.5 g/L of biotinylated recombinant CD38 and magnetic beads successfully extracted the DARA with a magnetic stand and completely eliminated the IgG kappa component on sIFE gel with all tested concentrations of recombinant CD38.

    Journal: Diagnostics

    Article Title: Complete Depletion of Daratumumab Interference in Serum Samples from Plasma Cell Myeloma Patients Improves the Detection of Endogenous M-Proteins in a Preliminary Study

    doi: 10.3390/diagnostics10040219

    Figure Lengend Snippet: Pooled patient sera with no endogenous monoclonal immunoglobulin protein (M-protein) spiked with DARA resulted in a clear IgG (G) kappa (K) M-protein (indicated by arrows) on serum immunofixation (sIFE) gel. Incubation of DARA-spiked serum with 0.125–0.5 g/L of biotinylated recombinant CD38 and magnetic beads successfully extracted the DARA with a magnetic stand and completely eliminated the IgG kappa component on sIFE gel with all tested concentrations of recombinant CD38.

    Article Snippet: The spiked sera aliquots were then supplemented with 0.125–0.5 g/L of biotinylated recombinant, human CD38 (Sino Biological, city, country, Catalog number: 10818-H08H-B) and incubated for ten minutes on an end-over-end tube rotator at room temperature.

    Techniques: Incubation, Recombinant, Magnetic Beads

    A representative of plasma cell myeloma (PCM) patient sera with endogenous IgG (G) kappa (K) M-protein spiked with DARA resulted in a clear IgG kappa M-protein (indicated by arrows) on sIFE gel. Incubation of DARA spiked PCM serum with 0.125 g/L of biotinylated recombinant CD38 and magnetic beads successfully extracted the DARA with a magnetic stand and completely eliminated the IgG kappa component on sIFE gel with all tested concentrations of recombinant CD38. CD38/DARA complex does not affect the endogenous M-protein migration.

    Journal: Diagnostics

    Article Title: Complete Depletion of Daratumumab Interference in Serum Samples from Plasma Cell Myeloma Patients Improves the Detection of Endogenous M-Proteins in a Preliminary Study

    doi: 10.3390/diagnostics10040219

    Figure Lengend Snippet: A representative of plasma cell myeloma (PCM) patient sera with endogenous IgG (G) kappa (K) M-protein spiked with DARA resulted in a clear IgG kappa M-protein (indicated by arrows) on sIFE gel. Incubation of DARA spiked PCM serum with 0.125 g/L of biotinylated recombinant CD38 and magnetic beads successfully extracted the DARA with a magnetic stand and completely eliminated the IgG kappa component on sIFE gel with all tested concentrations of recombinant CD38. CD38/DARA complex does not affect the endogenous M-protein migration.

    Article Snippet: The spiked sera aliquots were then supplemented with 0.125–0.5 g/L of biotinylated recombinant, human CD38 (Sino Biological, city, country, Catalog number: 10818-H08H-B) and incubated for ten minutes on an end-over-end tube rotator at room temperature.

    Techniques: Incubation, Recombinant, Magnetic Beads, Migration

    A representative patient sample is shown for illustration of the impact of pre-treatment and efficiency of recombinant biotinylated CD38 tested in sera from patients with PCM (IgG kappa indicated by arrows) who were receiving DARA as a therapy. CD38 ligand antibody completely removed the IgG kappa band associated with DARA.

    Journal: Diagnostics

    Article Title: Complete Depletion of Daratumumab Interference in Serum Samples from Plasma Cell Myeloma Patients Improves the Detection of Endogenous M-Proteins in a Preliminary Study

    doi: 10.3390/diagnostics10040219

    Figure Lengend Snippet: A representative patient sample is shown for illustration of the impact of pre-treatment and efficiency of recombinant biotinylated CD38 tested in sera from patients with PCM (IgG kappa indicated by arrows) who were receiving DARA as a therapy. CD38 ligand antibody completely removed the IgG kappa band associated with DARA.

    Article Snippet: The spiked sera aliquots were then supplemented with 0.125–0.5 g/L of biotinylated recombinant, human CD38 (Sino Biological, city, country, Catalog number: 10818-H08H-B) and incubated for ten minutes on an end-over-end tube rotator at room temperature.

    Techniques: Recombinant

    514G3 antibody binds to WT SpA with specificity and picomolar affinity. A. Antigen binding profiles of 514G3 captured on an active flow cell on a Biacore 3000. The colored lines on the sensogram represent the recorded binding response signals at different antigen concentrations from 2-0nM, and the overlaid black lines represent the fitted curves. Binding of SpA to 514G3 was monitored in real time to obtain on (ka) and off (kd) rates. The equilibrium constant (K D ) was calculated from the observed Ka and Kd and found to be 46.7pM. B. A graph showing the relative binding of 514G3 to SpA, MabSelect and MabSelect SuRe ligands. The three different SpA domain variants were coated on the wells of an ELISA plate, and then probed with biotinylated 514G3 or VH3/IgG3-k isotype control at 4μg/ml, and detected with streptavidin-HRP. The graph shows OD at 450nm on the y-axis and the different SpA domain variants on the X-axis. The error bars show the standard deviation from three replicates.

    Journal: PLoS ONE

    Article Title: A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia

    doi: 10.1371/journal.pone.0190537

    Figure Lengend Snippet: 514G3 antibody binds to WT SpA with specificity and picomolar affinity. A. Antigen binding profiles of 514G3 captured on an active flow cell on a Biacore 3000. The colored lines on the sensogram represent the recorded binding response signals at different antigen concentrations from 2-0nM, and the overlaid black lines represent the fitted curves. Binding of SpA to 514G3 was monitored in real time to obtain on (ka) and off (kd) rates. The equilibrium constant (K D ) was calculated from the observed Ka and Kd and found to be 46.7pM. B. A graph showing the relative binding of 514G3 to SpA, MabSelect and MabSelect SuRe ligands. The three different SpA domain variants were coated on the wells of an ELISA plate, and then probed with biotinylated 514G3 or VH3/IgG3-k isotype control at 4μg/ml, and detected with streptavidin-HRP. The graph shows OD at 450nm on the y-axis and the different SpA domain variants on the X-axis. The error bars show the standard deviation from three replicates.

    Article Snippet: Affinity selection of phage library with WT SpA Streptavidin conjugated Dynabeads (M-280 Streptavidin, ThermoFisher Scientific, 11205D) were coated with biotinylated SpA (Sigma-Aldrich, P6031).

    Techniques: Binding Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

    In vitro characterization of 514G3. A. 514G3 binds to S . aureus and leaves the Fc region exposed for FcγR1A receptor binding: 514G3 or VH3/IgG3-k isotype control was incubated with S . aureus cells for 15 min followed by the addition of biotin labeled FcγR1A. The figures show the overlay of median fluorescence intensities generated by the labeled antibodies or the Fcϒ receptor. There was a specific binding of 514G3 to the S . aureus cells within 15 min (left panel); and FcγR1A receptor could subsequently bind to the Fc region of 514G3 antibody bound to the surface of S . aureus (right panel). The right panel also shows two controls where S . aureus cells were incubated with biotinylated FcγR1A along with streptavidin-APC (light green trace) or streptavidin-APC only (dark green trace) to show that the high background is due to streptavidin-APC. The cells were analyzed on a BD Accuri and the data was analyzed using FlowJo 10.0.8. B. 514G3 is capable of binding to S . aureus in the presence of pooled human IgG. S . aureus was pre-incubated with 5mg of pooled human immunoglobulins at 37°C, before the addition of 100 μg/ml of 514G3 or the VH3/IgG3-k isotype control labeled with APC. There was an increase in the binding of 514G3 to S . aureus cell surface over the 2.5 hours tested. However, the signal with VH3/IgG3-k isotype remained the same over the time points tested. The graph plots the signal in the FL4 (APC) channel versus the total cell count. The median fluorescence intensity values are indicated in the column Median:FL4. The cells were analyzed on a BD Accuri and the data was analyzed using FlowJo 10.0.8, and the data is representative from multiple experimental repeats.

    Journal: PLoS ONE

    Article Title: A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia

    doi: 10.1371/journal.pone.0190537

    Figure Lengend Snippet: In vitro characterization of 514G3. A. 514G3 binds to S . aureus and leaves the Fc region exposed for FcγR1A receptor binding: 514G3 or VH3/IgG3-k isotype control was incubated with S . aureus cells for 15 min followed by the addition of biotin labeled FcγR1A. The figures show the overlay of median fluorescence intensities generated by the labeled antibodies or the Fcϒ receptor. There was a specific binding of 514G3 to the S . aureus cells within 15 min (left panel); and FcγR1A receptor could subsequently bind to the Fc region of 514G3 antibody bound to the surface of S . aureus (right panel). The right panel also shows two controls where S . aureus cells were incubated with biotinylated FcγR1A along with streptavidin-APC (light green trace) or streptavidin-APC only (dark green trace) to show that the high background is due to streptavidin-APC. The cells were analyzed on a BD Accuri and the data was analyzed using FlowJo 10.0.8. B. 514G3 is capable of binding to S . aureus in the presence of pooled human IgG. S . aureus was pre-incubated with 5mg of pooled human immunoglobulins at 37°C, before the addition of 100 μg/ml of 514G3 or the VH3/IgG3-k isotype control labeled with APC. There was an increase in the binding of 514G3 to S . aureus cell surface over the 2.5 hours tested. However, the signal with VH3/IgG3-k isotype remained the same over the time points tested. The graph plots the signal in the FL4 (APC) channel versus the total cell count. The median fluorescence intensity values are indicated in the column Median:FL4. The cells were analyzed on a BD Accuri and the data was analyzed using FlowJo 10.0.8, and the data is representative from multiple experimental repeats.

    Article Snippet: Affinity selection of phage library with WT SpA Streptavidin conjugated Dynabeads (M-280 Streptavidin, ThermoFisher Scientific, 11205D) were coated with biotinylated SpA (Sigma-Aldrich, P6031).

    Techniques: In Vitro, Binding Assay, Incubation, Labeling, Fluorescence, Generated, Cell Counting