surface plasmon resonance spr measurement  (Biacore)

Journal: International journal of molecular sciences

Article Title: Efficient Design of Affilin ® Protein Binders for HER3.

doi: 10.3390/ijms26104683

Figure Lengend Snippet: Figure 3. General workflow to generate and identify specific Affilin® proteins for a given target. The process begins by selecting a target protein, followed by a phage display screening using highly diverse Affilin® libraries against the target. Positive binding phage pools are then subjected to a high-throughput screening (HTS), where up to 15,000 single clones can be analyzed, each representing a unique Affilin® protein variant. Specific binding variants are nominated as hit. Subsequently, hits undergo a preliminary selection applying a µ-scale expression and purification in a 96-well format including a fast small-scale purification by using PhyNexus columns. Binding affinity is tested with SPR and fluorescence activated cell sorting (FACS) measuring techniques. The most promising Affilin® variants selected for an upscaling process in the protein expression and purification using, e.g., gel filtration and affinity chromatography systems. Highly purified samples then undergo a comprehensive protein analysis including concentration-dependent SPR, FACS, serum stability assays and differential scanning fluorimetry (DSF).

Article Snippet: Lab scale purified Affilin® proteins were used in a concentration-dependent manner for surface plasmon resonance (SPR) measurement, which was performed on a Sierra SPR-32 (Bruker, Billerica, MA, USA) with HER3-Fc immobilized on a Protein A-coated chip.

Techniques: Binding Assay, High Throughput Screening Assay, Clone Assay, Variant Assay, Selection, Expressing, Purification, Fluorescence, FACS, Filtration, Affinity Chromatography, Concentration Assay

Journal: International journal of molecular sciences

Article Title: Efficient Design of Affilin ® Protein Binders for HER3.

doi: 10.3390/ijms26104683

Figure Lengend Snippet: Figure 4. Concentration-dependent SPR data of lab scale purified Exp-1 to Exp-4. SPR was performed on a Sierra SPR-32 (Bruker) with a Protein A-coated chip on which the HER3-Fc was immobilized. The concentration of the curves is given in the upper right corner of each individual graph. Below the concentrations, the measured kinetic values are given. (A) SPR data for Exp-1. (B) SPR data for Exp-2. (C) SPR data for Exp-3. (D) SPR data for Exp-4.

Article Snippet: Lab scale purified Affilin® proteins were used in a concentration-dependent manner for surface plasmon resonance (SPR) measurement, which was performed on a Sierra SPR-32 (Bruker, Billerica, MA, USA) with HER3-Fc immobilized on a Protein A-coated chip.

Techniques: Concentration Assay, Purification

Journal: International journal of molecular sciences

Article Title: Efficient Design of Affilin ® Protein Binders for HER3.

doi: 10.3390/ijms26104683

Figure Lengend Snippet: Figure 6. Concentration-dependent SPR binding curves of different matured, lab scale purified Affilin® proteins. Mat-ExpComp variants were maturated computationally and show the best binding affinities against HER3-Fc. The Exp-MatExp variants were experimentally maturated and show very similar affinities towards HER3-Fc. SPR was performed on a Sierra SPR-32 (Bruker) with a Protein A-coated chip on which the HER3-Fc was immobilized.

Article Snippet: Lab scale purified Affilin® proteins were used in a concentration-dependent manner for surface plasmon resonance (SPR) measurement, which was performed on a Sierra SPR-32 (Bruker, Billerica, MA, USA) with HER3-Fc immobilized on a Protein A-coated chip.

Techniques: Concentration Assay, Binding Assay, Purification