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Biacore biosensor-based surface plasmon resonance (spr) analysis
Biosensor Based Surface Plasmon Resonance (Spr) Analysis, supplied by Biacore, 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/product/spr+biosensor+analysis/biacore+3000+instrument/pmc06300143-481-7-12
Average 90 stars, based on 1 article reviews

biosensor-based surface plasmon resonance (spr) analysis - by Bioz Stars, 2026-10

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Article Title: Methods for treating heterotopic ossification
Article Snippet: Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the BiacoreTM binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunohistochemistry.

Article Title: ALK7 binding proteins and uses thereof
Article Snippet: In particular embodiments, the variant ALK7-binding protein has at least one characteristic selected from: (a) decreases the formation of a complex containing ALK7, a type II receptor (e.g., ActRIIA or ActRIIB), and one or more TGF-beta superfamily ligands (e.g., activin B, activin AB, Nodal, GDF1, GDF3 and/or GDF8) on the surface of cells expressing ALK7 and the ActRII receptor in the presence of the one or more TGF-beta superfamily ligands; (b) competes with one or more type II receptors for binding to ALK7; (c) competes with one or more TGF-beta superfamily ligands (e.g., activin B, activin AB, Nodal, GDF1, GDF3 and/or GDF8) for binding to ALK7; (d) decreases the phosphorylation of ALK7 in cells expressing ALK7 and a type II receptor (e.g., ActRIIA or ActRIIB) in the presence of one or more TGF-beta super family ligands (e.g., GDF1, GDF3, GDF8, activin B, activin AB, and/or Nodal); (e) decreases the phosphorylation of Smads (e.g., Smad2 and/or Smad3) in cells expressing ALK7 and a type II receptor (e.g., ActRIIA and/or ActRIIB) in the presence of one or more TGF-beta ligands (e.g., GDF1, GDF3, GDF8, activin B, activin AB, and/or Nodal); (f) binds to ALK7 with a KD of ≤1 nM and ≥1 pM (e.g., as determined by BIACORE® analysis), and (g) decreases the formation of a complex containing ALK7, a co-receptor (e.g., cripto and/or cryptic), and one or more TGF-beta superfamily ligands (e.g., Nodal).

Article Title: ALK7 binding proteins and uses thereof
Article Snippet: In some embodiments, an ALK7-binding protein binds to ALK7 with a KD of <1 nM and ≥1 pM (e.g., as determined by BIACORE® analysis).

Binding Assay:

Article Title: ALK7 binding proteins and uses thereof
Article Snippet: The BIACORE® assay described above is an exemplary assay used to determine if two ALK7-binding proteins such as anti-ALK7 antibodies compete with/block each other for binding ALK7. .. On rare occasions, particular ALK7-binding proteins may not bind to ALK7-Fc coupled via anti-Fc IgG to a CM5 BIACORE® chip (this might occur when the relevant binding site on ALK7 is masked or destroyed by ALK7 linkage to Fc). ..

Article Title: Anti-CD40 antibodies and uses thereof
Article Snippet: 5.4 Quantitative Analysis of NK003 Dynamics and Affinity by Surface Plasmon Resonance Biacore T200 (GE Healthcare) was used to detect the affinity of the antibodies NK003, NK004. .. Data processing was performed using BIAevaluation software S200, the necessary software of Biacore T200 instrument and binding constants (Ka), dissociation constants (Kd), and equilibrium dissociation constants (KD) were calculated. .. The antigen to be tested, i.e. human CD40 recombinant protein (Acrobiosystems, CD 0-H5228) was diluted gradiently with a Running buffer (HBS-EP+, GE) at concentrations of 2 μM, 1 μM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM.

Article Title: ALK7 binding proteins and uses thereof
Article Snippet: .. Likewise, known assays are available for readily selecting anti-ALK7-antibodies displaying desirable features (e.g., assays for determining binding affinity to ALK7; cross-blocking assays such as the BIACORE®-based human ALK7-binding protein competition binding assays described herein). ..

SPR Assay:

Article Title: NKp46 binding agents
Article Snippet: .. Competition and Affinity Study by Surface Plasmon Resonance (SPR) Biacore T100 General Procedure and Reagents SPR measurements were performed on a Biacore T100 apparatus (Biacore GE Healthcare) at 25° C. In all Biacore experiments HBS-EP+(Biacore GE Healthcare) and NaOH 10 mM NaCl 500 mM served as running buffer and regeneration buffer respectively. .. After two washes in staining buffer, cells incubated with anti-NKp46 antibodies were stained for 30 min at 4° C. with mouse anti-human IgG1-PE monoclonal antibodies from Miltenyi (dil. 1/10).

Article Title: Antibodies binding to phospho-tau comprising phosphorylated Ser396 and Ser404 and methods of detecting thereof
Article Snippet: In accordance with the present invention, the antibody-based molecule of the present invention binds to its Tau epitope with an affinity corresponding to a KD of about 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a Biacore 3000 instrument (preferably using the antibody as the ligand and the antigen as the analyte). .. In accordance with the present invention, the antibody-based molecule of the present invention binds to its Tau epitope with an affinity corresponding to a KD of about 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a Biacore 3000 instrument (preferably using the antibody as the ligand and the antigen as the analyte). ..

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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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biosensor-based surface plasmon resonance (spr) analysis (Biacore)

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