Nhs peg4 biotin

SPR was performed using a Biacore T200 (Cytiva) and Series S Sensorchip SA (Cytiva, BR100531) at the temperature of 25 °C. A solution of PBS, 0.005% TWEEN20 (Sigma, P9416), 0.5 mM TCEP (Nacalai Tesque, 06342-21), and 2% DMSO (Sigma, 276855) was filtered with a 0.22-μm filter and used as the running buffer. Kinetic measurements were carried out at a flow rate of 30 μL/min, and the association and dissociation phases were monitored for 120 s and 120–300 s, respectively. The G9a SET domain was biotinylated by using NHS-PEG4-Biotin (Thermo, 39259), and captured to flow cells 2, 3, and 4 with immobilization levels of ~2300 RU. No specific treatment was applied to the reference surface (flow cell 1). To prevent oxidation of the immobilized protein, a solution containing 1 μM ZnCl₂ and 5 mM TCEP was injected at a flow rate of 10 μL/min for 60 s before each set of kinetic measurements. The extra wash command was executed after each measurement cycle with 50% DMSO aqueous solution. To correct for bulk responses, solvent correction was performed once after all kinetics runs by using running buffer containing 1–3% DMSO. The sensorgrams obtained from the three flow cells were analyzed individually by Biacore T200 Evaluation Software (Cytiva). The dissociation constant (K_D) and binding kinetic parameters were calculated by a curve fit to a built-in 1:1 binding model.

SPR studies were performed on a Biacore T200 instrument (Cytiva). For analysis of OX40 binding by Anticalin proteins, human OX40-His was immobilized at 2.5 µg/ml in 10 mM sodium acetate pH 5.0 on CM5 Series S sensor chips to ∼140 RU using Amine Coupling Kit (Cytiva). HSA (Sigma, #A3782), MSA (Albumin Bioscience), trastuzumab (Roche) and mouse IgG (Sigma, #I5381) were randomly biotinylated using NHS–PEG4–biotin (ThermoFischer Scientific) and immobilized with Biotin CAPture Kit, Series S (Cytiva). Kinetic measurements were performed by injecting serial dilutions of Anticalin proteins in running buffer HBS-EP+, pH 7.4 at a flow rate of 30 µl/min and a temperature of 25°C. For measurements in presence of ABD or IgBD interaction partners, analytes were diluted in HBS-EP+ buffer containing 1.5 µM HSA or trastuzumab. The CM5 sensor chip was regenerated with 10 mM glycine-HCl, pH 2.5 for 60 s. All binding curves were referenced to HBS–EP+ sample and reference flow cell values were subtracted. Binding kinetics parameters were determined using the 1:1 Langmuir binding model or steady state affinity provided by the Biacore T200 Evaluation Software, version 3.0.

The antibody microarrays were prepared as previously described [15 (link)]. Ten microliters of serum was diluted 1:10 with phosphate buffered saline (PBS; pH 7.4) and then labeled with NHS-PEG4-Biotin (Thermo Fisher Scientific, MA, USA). After removing the excess biotin molecules, the biotinylated serum was diluted with 400 μL of 5% milk (w/v) and then incubated with antibody microarrays that were blocked for 1 h at room temperature with 500 μL of 5% milk (w/v). Subsequently, the antibody microarrays were washed with PBS containing 0.05% (w/v) Tween 20 (PBST). The bound proteins on microarrays were detected by incubating with 2 µg/mL streptavidin–phycoerythrin (PE) (Jackson Immunoresearch, USA) for 1 h at room temperature. After washing and drying, the microarrays were scanned using the GenePix 4300A microarray scanner.

Cells were plated on 75 µg/ml collagen I-coated tissue culture plastic at confluence and allowed to attach overnight. Cells were quickly washed in phosphate-buffered saline (PBS) three times, and cells were dissociated with a non-enzymatic dissociation buffer (3 mM EDTA, 2% glycerol, 15 mM sodium citrate in PBS, pH 7.4.) and resuspended in 1% BSA in PBS. Cells were washed two times in PBS containing Ca²⁺ and Mg²⁺ (PBS-2⁺; Gibco 14040117) and treated with 5 mM NHS-PEG4-Biotin (Thermo Fisher Scientific, #A39259) for surface labeling or 5 mM BS³ (Thermo Fisher Scientific, #PIA39266) in PBS-2⁺ medium for 30 min at 4°C. Cell suspensions were quenched with 20 mM Tris in PBS-2⁺ for 15 min and washed with 20 mM Tris in PBS-2⁺ twice to fully quench crosslinking reactions. Cells were then washed three times in PBS-2⁺ and lysed in denaturing lysis buffer (RIPA lysis buffer plus 0.5% SDS). Biotin-labeled cells were subjected to immunoprecipitation with streptavidin beads (Sigma-Aldrich; S1638) or anti-DDR2 antibodies and were immunoblotted as described above. BS³ crosslinking experiments were subjected to SDS–PAGE on 6% polyacrylamide gels and immunoblotted as described above.