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Biacore 3000 optical biosensor
3000 Optical Biosensor, supplied by Biacore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/spr+biosensor+analysis/3000+biacore+instrument/pmc03774728-45-8-7
Average 86 stars, based on 1 article reviews

3000 optical biosensor - by Bioz Stars, 2026-11

86/100 stars

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Binding Assay:

Article Title: High affinity engineered T-cell receptors targeting cmv infected cells
Article Snippet: Data were analyzed with Graphpad Prism 5. .. Dynamic and equilibrium binding kinetics were obtained using a BIAcore 3000 instrument. .. The purified TCR-Fc was immobilized on a CM5 sensor chip (GE Healthcare) via EDC/NHS coupling using a sodium acetate buffer at pH 4.0 for a total of 2000-5000 response units, with a blank flow cell used as the reference channel.

Article Title: Broad Neutralizing Activity of Monoclonal Antibodies Against the Omicron Variants Isolated From Patients With Early Severe Acute Respiratory Syndrome Coronavirus-2.
Article Snippet: .. For SPR, binding kinetics between antibodies and S1 or RBD variants were assessed on a Biacore 3000 system. ..

Article Title: Humanized or chimeric CD3 antibodies
Article Snippet: .. The term “binding” as used herein, refers to the binding of an antibody to a predetermined antigen or target to which binding typically is with an affinity corresponding to a KD of about 10−6 M or less, e.g. 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−8 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. ..

SPR Assay:

Article Title: Chlorotoxin does not target matrix metalloproteinase-2 in glioblastoma
Article Snippet: Surface plasmon resonance (SPR) reagents, plates, and the NTA chip were purchased from GE Healthcare Life Sciences, now Cytiva (Marlborough, MA, USA). .. Measurements were performed using the Biacore 8000 SPR System (Marlborough, MA. ..

Article Title: Antibodies and chimeric antigen receptors that target TACI
Article Snippet: .. Typically, the antibodies bind to TACI with a dissociation equilibrium constant (KD) of less than about 10−6 M, such as less than approximately 10−7 M, 10−8 M, 10−9 M, or 10−10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 3000 instrument using recombinant TACI as the analyte and the antibody as the ligand. ..

Article Title: Heterodimeric proteins and methods for producing and purifying them
Article Snippet: .. The bispecific antibody was also able to simultaneously bind antigens A and B. Antigens C and D A Biacore 3000 SPR biosensor instrument was also used for this analysis. .. The (antigen D)-hFc was coupled to a Biacore CM5 sensor chip surface using an amine-coupling procedure.

Article Title: Antibodies and chimeric antigen receptors that target TACI
Article Snippet: .. For example, a specifically antibody may bind to its target with an affinity (KD) of approximately less than 10−7 M, such as approximately less than 10−8 M, 10−9 M or 10−10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 3000 instrument, which can be performed, for example, using recombinant TACI as the analyte and the antibody as the ligand. ..

Article Title: Antigen-binding molecules that bind CD38 and/or CD28, and uses thereof
Article Snippet: .. For CD38 kinetic determinations, SPR experiments were performed using a Biacore 3000 instrument at 25° C. Antibodies were captured for 37 seconds at a flow rate of 8 uL/min on a CM5 anti-humanFc-(bsAb2567)-coupled surface. ..

Recombinant:

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Chemical modification of lysine residues in the FVIII light chain abolishes the interaction with LRP1 cluster II. Association and dissociation of LRP1 cluster II to FVIII light chain were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain was bound to the anti-C2 antibody at a density of 17 fmol/mm−2. To study the contribution of lysine residues on the interaction between LRP1 cluster II and the FVIII light chain, lysine residues were modified by passing over 50 mm sulfo-NHS acetate or sulfo-NHS biotin (Thermo Fisher Scientific) for 10 min at 25 °C with a flow rate of 20 μl/min. A and B, LRP1 cluster II (0.2–200 nm) (A) and anti-a3 antibody CLB-CAg69 (0.1–100 nm) (B) were passed over the FVIII light chain in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain was corrected for binding in the absence of FVIII. The response at 235 s after association is plotted as a function of the concentration.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Chemical modification of lysine residues in the FVIII light chain abolishes the interaction with LRP1 cluster II. Association and dissociation of LRP1 cluster II to FVIII light chain were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain was bound to the anti-C2 antibody at a density of 17 fmol/mm−2. To study the contribution of lysine residues on the interaction between LRP1 cluster II and the FVIII light chain, lysine residues were modified by passing over 50 mm sulfo-NHS acetate or sulfo-NHS biotin (Thermo Fisher Scientific) for 10 min at 25 °C with a flow rate of 20 μl/min. A and B, LRP1 cluster II (0.2–200 nm) (A) and anti-a3 antibody CLB-CAg69 (0.1–100 nm) (B) were passed over the FVIII light chain in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain was corrected for binding in the absence of FVIII. The response at 235 s after association is plotted as a function of the concentration.

Article Snippet: Surface Plasmon Resonance Analysis Association and dissociation of LRP1 cluster II to FVIII light chain and FVIIIdB variants were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB, Uppsala, Sweden).

Techniques: Modification, Concentration Assay, Binding Assay

Library of lysine to arginine replacements in the FVIII light chain identifies the contribution of multiple lysine residues in the interaction with LRP1 cluster II. Association and dissociation of LRP1 cluster II to FVIII light chain variants were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm−2. LRP1 cluster II (0.2–200 nm) was passed over the FVIII light chain variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model. A, representative experiment for the interaction between LRP1 cluster II and wild type FVIII light chain. B, on each SPR sensor chip, we included a control channel (only CLB-EL14 IgG4), wild type FVIII light chain, and two FVIII light chain variants. Therefore, wild type FVIII light chain was analyzed multiple times (n = 14) C and D, representative experiments for variants. E, the KD for LRP1 cluster II for the FVIII light chain variants was compared with the KD of wild type FVIII light chain (n = 14, degrees of freedom = 13, *, p < 0.10, t value = 2.16, ***, p <0.001, t value = 4.22) using a two-tailed Student's t test. Error bars indicate ± S.D.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Library of lysine to arginine replacements in the FVIII light chain identifies the contribution of multiple lysine residues in the interaction with LRP1 cluster II. Association and dissociation of LRP1 cluster II to FVIII light chain variants were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm−2. LRP1 cluster II (0.2–200 nm) was passed over the FVIII light chain variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model. A, representative experiment for the interaction between LRP1 cluster II and wild type FVIII light chain. B, on each SPR sensor chip, we included a control channel (only CLB-EL14 IgG4), wild type FVIII light chain, and two FVIII light chain variants. Therefore, wild type FVIII light chain was analyzed multiple times (n = 14) C and D, representative experiments for variants. E, the KD for LRP1 cluster II for the FVIII light chain variants was compared with the KD of wild type FVIII light chain (n = 14, degrees of freedom = 13, *, p < 0.10, t value = 2.16, ***, p <0.001, t value = 4.22) using a two-tailed Student's t test. Error bars indicate ± S.D.

Techniques: Concentration Assay, Binding Assay, Control, Two Tailed Test

Lysine to alanine and lysine to glutamic acid replacements at position 1693, 1827, 1967, 2065, and 2092. Association and dissociation of LRP1 cluster II to FVIII light chain variants Lys1693, Lys1827, Lys1967, Lys2065, and Lys2092 were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm−2. LRP1 cluster II (0.2–200 nm) was passed over the FVIII light chain variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Lysine to alanine and lysine to glutamic acid replacements at position 1693, 1827, 1967, 2065, and 2092. Association and dissociation of LRP1 cluster II to FVIII light chain variants Lys1693, Lys1827, Lys1967, Lys2065, and Lys2092 were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm−2. LRP1 cluster II (0.2–200 nm) was passed over the FVIII light chain variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model.

Techniques: Concentration Assay, Binding Assay

Effect of replacement of lysine residues by positively charged arginine, uncharged alanine, or negatively charged glutamic acid on the interaction between the FVIII light chain and LRP1 cluster II Association and dissociation of LRP1 cluster II to FVIII light chain variants Lys-1693, Lys-1827, Lys-1967, Lys-2065, and Lys-2092 was assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB, Uppsala, Sweden). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm −2 ) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm −2 . LRP1 cluster II (0.2–200 n m ) was passed over the FVIII light chain variants in a buffer containing 150 m m NaCl, 5 m m CaCl 2 , 0.005% (v/v) Tween 20, and 20 m m Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model. ND, not determined.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Effect of replacement of lysine residues by positively charged arginine, uncharged alanine, or negatively charged glutamic acid on the interaction between the FVIII light chain and LRP1 cluster II Association and dissociation of LRP1 cluster II to FVIII light chain variants Lys-1693, Lys-1827, Lys-1967, Lys-2065, and Lys-2092 was assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB, Uppsala, Sweden). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm −2 ) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm −2 . LRP1 cluster II (0.2–200 n m ) was passed over the FVIII light chain variants in a buffer containing 150 m m NaCl, 5 m m CaCl 2 , 0.005% (v/v) Tween 20, and 20 m m Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model. ND, not determined.

Techniques: Concentration Assay, Binding Assay

Combined lysine replacements have an additive effect on LRP1 cluster II interaction. Association and dissociation of LRP1 cluster II to FVIII light chain variants carrying replacements at positions Lys1693, Lys1827, and Lys1967 (A3), Lys2065 and Lys2092 (C1), and Lys1693, Lys1827, Lys1967, Lys2065, and Lys2092 (A3C1) were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm−2. LRP1 cluster II (0.2–200 nm) was passed over the FVIII light chain variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Combined lysine replacements have an additive effect on LRP1 cluster II interaction. Association and dissociation of LRP1 cluster II to FVIII light chain variants carrying replacements at positions Lys1693, Lys1827, and Lys1967 (A3), Lys2065 and Lys2092 (C1), and Lys1693, Lys1827, Lys1967, Lys2065, and Lys2092 (A3C1) were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm−2) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm−2. LRP1 cluster II (0.2–200 nm) was passed over the FVIII light chain variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model.

Techniques: Concentration Assay, Binding Assay

Effect of combining lysine replacements on the interaction between the FVIII light chain and LRP1 cluster II Association and dissociation of LRP1 cluster II to FVIII light chain variants carrying replacements at positions Lys-1693, Lys-1827, and Lys-1967 (A3), Lys-2065 and Lys-2092 (C1), and Lys-1693, Lys-1827, Lys-1967, Lys-2065, and Lys-2092 (A3C1) was assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB, Uppsala, Sweden). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm −2 ) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm −2 . LRP1 cluster II (0.2–200 n m ) was passed over the FVIII light chain variants in a buffer containing 150 m m NaCl, 5 m m CaCl 2 , 0.005% (v/v) Tween 20, and 20 m m Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Effect of combining lysine replacements on the interaction between the FVIII light chain and LRP1 cluster II Association and dissociation of LRP1 cluster II to FVIII light chain variants carrying replacements at positions Lys-1693, Lys-1827, and Lys-1967 (A3), Lys-2065 and Lys-2092 (C1), and Lys-1693, Lys-1827, Lys-1967, Lys-2065, and Lys-2092 (A3C1) was assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB, Uppsala, Sweden). The anti-C2 antibody CLB-EL14 IgG4 (26 fmol/mm −2 ) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIII light chain variants were bound to the anti-C2 antibody at a density of 17 fmol/mm −2 . LRP1 cluster II (0.2–200 n m ) was passed over the FVIII light chain variants in a buffer containing 150 m m NaCl, 5 m m CaCl 2 , 0.005% (v/v) Tween 20, and 20 m m Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIII light chain variants was corrected for binding in absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model.

Techniques: Concentration Assay, Binding Assay

Binding of FVIII light chain variants to full-length LRP1. A, association and dissociation of FVIII light chain variants to full-length LRP1 were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). LRP1 (Biomac) was coupled directly on a CM5 chip according to the manufacturer's instructions at three different densities (15, 18, and 21 fmol/mm−2). FVIII light chain variants (1–25 μg/ml based on Bradford analysis) were passed over the immobilized LRP1 in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of FVIII using the same buffer containing 1 m NaCl. Binding to LRP1 was corrected for binding in the absence of LRP1. Shown are the SPR curves for the channel on which 21 fmol/mm−2 LRP1 was coupled. B, The response units at time point 235 s were plotted as a function of the concentration for all three LRP1 surface densities.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Binding of FVIII light chain variants to full-length LRP1. A, association and dissociation of FVIII light chain variants to full-length LRP1 were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). LRP1 (Biomac) was coupled directly on a CM5 chip according to the manufacturer's instructions at three different densities (15, 18, and 21 fmol/mm−2). FVIII light chain variants (1–25 μg/ml based on Bradford analysis) were passed over the immobilized LRP1 in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of FVIII using the same buffer containing 1 m NaCl. Binding to LRP1 was corrected for binding in the absence of LRP1. Shown are the SPR curves for the channel on which 21 fmol/mm−2 LRP1 was coupled. B, The response units at time point 235 s were plotted as a function of the concentration for all three LRP1 surface densities.

Techniques: Binding Assay, Concentration Assay

Binding of FVIIIdB variants to LRP1 cluster II. A, association and dissociation of LRP1 cluster II to FVIIIdB variants were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (39 fmol/mm−2 for FVIIIdB variants) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIIIdB variants were bound to the anti-C2 antibody at a density of 9 fmol/mm−2. LRP1 cluster II (0–200 nm) was passed over the FVIIIdB variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIIIdB variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model. B, the responses at equilibrium were fitted by non-linear regression using a standard hyperbola to generate KD values (GraphPad Prism 4 software). Error bars indicate ± S.D.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Binding of FVIIIdB variants to LRP1 cluster II. A, association and dissociation of LRP1 cluster II to FVIIIdB variants were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). The anti-C2 antibody CLB-EL14 IgG4 (39 fmol/mm−2 for FVIIIdB variants) was immobilized onto a CM5 sensor chip using the amine coupling method according to the manufacturer's instructions. Subsequently, FVIIIdB variants were bound to the anti-C2 antibody at a density of 9 fmol/mm−2. LRP1 cluster II (0–200 nm) was passed over the FVIIIdB variants in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of LRP1 cluster II using the same buffer containing 1 m NaCl. Binding to FVIIIdB variants was corrected for binding in the absence of FVIII. Binding data during the association phase were fitted in a one-phase exponential association model. B, the responses at equilibrium were fitted by non-linear regression using a standard hyperbola to generate KD values (GraphPad Prism 4 software). Error bars indicate ± S.D.

Techniques: Binding Assay, Concentration Assay, Software

Binding of FVIIIdB variants to full-length LRP1. A, association and dissociation of FVIIIdB variants to full-length LRP1 were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). LRP1 (Biomac) was coupled directly on a CM5 chip according to the manufacturer's instructions at three different densities (15, 18, and 21 fmol/mm−2). FVIIIdB variants (5–1000 units/ml based on chromogenic activity) were passed over the immobilized LRP1 in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of FVIII using the same buffer containing 1 m NaCl. Binding to LRP1 was corrected for binding in the absence of LRP1. Shown are the SPR curves for the channel on which 21 fmol/mm−2 LRP1 was coupled. B, the response units at time point 235 s were plotted as a function of the concentration for all three LRP1 surface densities.

Journal: The Journal of Biological Chemistry

Article Title: Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues ^♦

doi: 10.1074/jbc.M115.650911

Figure Lengend Snippet: Binding of FVIIIdB variants to full-length LRP1. A, association and dissociation of FVIIIdB variants to full-length LRP1 were assessed by SPR analysis employing a BIAcore 3000 biosensor (Biacore AB). LRP1 (Biomac) was coupled directly on a CM5 chip according to the manufacturer's instructions at three different densities (15, 18, and 21 fmol/mm−2). FVIIIdB variants (5–1000 units/ml based on chromogenic activity) were passed over the immobilized LRP1 in a buffer containing 150 mm NaCl, 5 mm CaCl2, 0.005% (v/v) Tween 20, and 20 mm Hepes (pH 7.4) at 25 °C with a flow rate of 20 μl/min. The sensor chip surface was regenerated three times after each concentration of FVIII using the same buffer containing 1 m NaCl. Binding to LRP1 was corrected for binding in the absence of LRP1. Shown are the SPR curves for the channel on which 21 fmol/mm−2 LRP1 was coupled. B, the response units at time point 235 s were plotted as a function of the concentration for all three LRP1 surface densities.

Techniques: Binding Assay, Activity Assay, Concentration Assay

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