Hbs n buffer

Surface plasmon resonance (SPR) measurements were performed on a Biacore T200 instrument equipped with CM5 sensor chip with ~4000 response units (RU) Clk4 (Abcam#AB204144), ~400 RU Clk1 and ~14,000 RU Clk2 (Abcam #AB63190) covalently immobilized with NHS/EDC to the surface. Additionally, SPR was performed on ~300 RU Clk1, 1000 RU Clk2 and 1000 RU Clk4 covalently attached to a CM5 Chip. For Clk1, Ni‐NTA SPR was used non‐covalently attaching His‐Labeled Clk1 at ~1800 RU for Clk1 binding to TG003 and for TG003 inhibition of PGC1α to Clk1 approximately ~1000 RU of PGC1α was non‐covalently attached to the chip. TG003 and PGC1α were titrated and flowed over these chips in 10× HBS‐N buffer from GE diluted in ultrapure water and filtered. Binding was expressed in relative response units (RU); the difference in response between the immobilized protein flow cell and the corresponding control subtracting blanks and utilizing a solvent control for DMSO generated from 0.5, 0.75, 1, 1.25 and 1.5 percent DMSO. TG003 was applied to chips containing Clk1,2 and 4 using 1:1 titrations and results exported from BiaEvaluate software into GraphPad prism (GraphPad Software). Saturation curves for PGC1α binding to Clk1 were fit using a specific binding equation with Hill slope, whereas all other SPR saturation curves were fit using a 1:1 specific binding model.

The dynamics of SINEUP-ILF3 interactions were characterized by SPR using a Biacore T100 instrument (GE Healthcare) as previously described in Patrucco et al. (36 (link)). The biotinylated invSINEB2 RNA was immobilized on streptavidin-coated sensor chips (Series S Sensor Chip SA; GE Healthcare). RNA was diluted to a final concentration of 1 μM in 10 mM HEPES and150 mM NaCl, pH 7.4 (HBS-N buffer, GE Healthcare), followed by heating at 80°C for 10 min and cooling to room temperature. The sample was then diluted 500-fold in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.025% surfactant P20; GE Healthcare) and injected over the sensor chip surface at 5 µl/min at 25°C to generate an ∼150 response unit.
GST-dsRBM2 was serially diluted in running buffer to the concentrations 300–3.7 nM and injected at 25°C at a flow rate of 30 µl/min for 2 min. Analysis were performed in duplicate, and any background signal from a streptavidin-only reference flow cell was subtracted from every data set.

SPR studies were performed using a Biacore T200 system (GE Healthcare). Recombinant ficolin-3 (R&D Systems) was immobilized on CM5 sensor chip (GE Healthcare Bio-Science AB) using amine coupling chemistry. Recombinant ficolin-3 was injected as a 50 μg/ml solution in 10 mM sodium acetate (pH 4.0) at a flow rate of 5 μl/min to a level of 15,300 resonance units (RU). The reference flow cell was immobilized with 12000 RU of BSA (Sigma-Aldrich). HBS-N buffer (GE Healthcare) supplemented with Ca²⁺ and Mg²⁺ ions (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% v/v surfactant P20, 5 mM MgCl₂, 5 mM CaCl₂) was used as a running buffer and sample buffer. Different concentrations of human IgG, IgA, and IgM (Sigma-Aldrich), recombinant MBL (R&D Systems) and O-PS 1200 were injected over the surface of immobilized Recombinant ficolin-3 and BSA (reference flow cell) at a flow rate of 30 μl/min for 150 s or 600 s (O-PS 1200); 0.5% SDS injected for 30 s was used as a regenerator in all SPR experiments. BSA or ethanolamine were used to optimize a reference surfaces for analytes IgM and IgA.

N-terminal His-tagged FabG from E. coli and C-terminal His-tagged FabG from Staphylococcus aureus were cloned into the NcoI and XhoI cloning sites of the vector pET28a, expressed in BL21 DE3 E. coli and further purified with Ni resin (Kristan et al., 2009 (link); Srinivas and Cronan, 2017 (link)). The binding interaction between tachyplesin III and FabG was analyzed by surface plasmon resonance (SPR) with a Biocore 3000 (Biocore, Piscataway, NJ, United States). Briefly, tachyplesin III was adsorbed onto a CM5 sensor chip using an amine-coupling kit (GE Healthcare, Little Chalfont, United Kingdom) to obtain approximately 1000 resonance units. For the analysis, different concentrations of FabG proteins in running buffer (HBS-N buffer, GE Healthcare) were injected at a flow rate of 20 μl/min for 6 min. The binding affinity of FabG for tachyplesin III was determined by BIAevaluation 3.0 software (Biacore) with a 1:1 Langmuir binding model for the kinetic calculation (Wei et al., 2013 (link)).