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Biacore spr biosensor analysis
Spr Biosensor 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/spr+biosensor+analysis/pm39234811-348-4-14
Average 90 stars, based on 1 article reviews

spr biosensor analysis - by Bioz Stars, 2026-10

90/100 stars

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SPR Assay:

Article Title: Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology
Article Snippet: .. SPR biosensor assays were performed on Biacore 3000 and Biacore S200 instruments equipped with the CM5 sensor chip (GE healthcare). .. For the immobilization of ActRIIA-Fc and ActRIIB-Fc, PBS supplied with 0.01% Surfactant P20 (GE healthcare) was used as the running buffer with the instrument temperature set at 22 °C.

Article Title: Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.
Article Snippet: .. Surface PlasmonResonance (SPR) Biosensor Assays.The SPR experiments were performed using a Biacore S200 instrument and Sensor Chip CM5 (Cytiva, Uppsala, Sweden) at 25 °C. .. Streptavidin (Sigma) was immobilized by a standard amine coupling procedure.

Article Title: Simulation Strategies for Characterizing Phosphodiesterase-5 Inhibitors in Botanical Dietary Supplements.
Article Snippet: A novel “Prediction and Confirmation” (PC) strategy was proposed for characterizing phosphodiesterase-5 inhibitor (PDE-5) derivatives in botanical dietary supplements (BDSs) for on-site detection.. Discovery Studio (DS) and density functional theory (DFT) calculations were used for the “Prediction” step in order to estimate PDE-5 derivative structures and theoretical Raman shifts without synthesizing the derivatives.. After 11 potentially bioactive sildenafil derivatives were acquired through DS, 32 common calculated Raman shifts were obtained through DFT.

Article Title: Predicting the intestinal absorption potential of hits and leads.
Article Snippet: Today’s pharma environment requires rapid and reliable methods of screening drug leads for intestinal permeability potential in the early stages of drug discovery.. Techniques using excised tissues, Caco-2 cells, artificial membranes, ‘in silico’ techniques and surface plasmon resonance (SPR)-based biosensors are critically examined in terms of their reliability, measurement criteria, throughput and utility in identifying potentially successful or unsuccessful drug molecules.

Article Title: The heat is on: thermodynamic analysis in fragment-based drug discovery.
Article Snippet: Thermodynamic analysis provides access to the determinants of binding affinity, enthalpy and entropy.. In fragment-based drug discovery (FBDD), thermodynamic analysis provides a powerful tool to discriminate fragments based on their potential for successful optimization.. The thermodynamic data generated by FBDD studies can in turn be used to better understand the forces that drive biomolecular interactions.

Article Title: Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody
Article Snippet: .. SPR biosensor analysis was conducted on a Biacore T100 platform with CM5 Series S sensor chip (GE Healthcare, Life Science, Milan, Italy). .. Amine Coupling kit and Mouse Antibody Capture kit (GE Healthcare, Life Science, Uppsala, Sweden) were respectively exploited for standard amine coupling and the two capturing mediated immobilization strategies.

Article Title: Compounds and methods
Article Snippet: .. The sixteen polypeptide conjugates were also characterized by SPR biosensor analysis (Biacore®, GE Healthcare), using a Biacore® 2000 instrument, Biacore® CM-5 chip and HBS-EP buffer pH 7.4 (Biacore®, GE Healthcare) with addition of 1% DMSO for the interaction studies. ..

Article Title: Targeting Dual Immune Checkpoints PD-L1 and HLA-G by Trispecific T Cell Engager for Treating Heterogeneous Lung Cancer.
Article Snippet: .. SPR Biosensor Analysis for Nb Binding Affinity: SPR biosensor analysis was conducted on a BIAcore T200 platform with CM5 series sensor chips or protein A chips (GE Healthcare, Illinois, USA) to determine NbTriTE binding affinity. ..

Biosensor Assay:

Comparison:

Cell Culture:

In Silico:

PAMPA Assay:

Binding Assay:

Image Search Results

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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spr biosensor analysis (Biacore)

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SPR Assay:

Biosensor Assay:

Comparison:

Cell Culture:

In Silico:

PAMPA Assay:

Binding Assay:

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