Gst capture kit

The binding of the SBD peptide to S100A4 was examined by Surface plasmon resonance (SPR) with a Biacore 3000 biosensor and GST Capture kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. Goat anti-GST antibody was immobilized on two flow cells of the sensor chip (CM-5) using a standard amine coupling procedure, and then recombinant GST-S100A4 or GST as a negative control was captured. The SBD peptide (1 mmol/l) in HBS-P running buffer (10 mmol/l Hepes, pH 7.4, 150 mmol/l NaCl, 0.005% P20) in the presence or absence of 1 mmol/l CaCl₂ was injected over two flow cells of the chip at a flow rate of 10 ml/minute at 25 °C for 5 minutes. The response from the control surface was subtracted, and the data plotted as Resonance Units (RU).

Anti-GST antibody was immobilized on a CM5 chip using a GST capture kit as described in the manufacturer’s instructions (GE Healthcare). 40 µl of 10 µg ml⁻¹ GST-FliJ or 10 µg ml⁻¹ of GST-FliJ(Δ13–24) were injected over the chip pre-equilibrated with a binding buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 m M EDTA, 0.005% Surfactant P20) at a flow rate of 20 µl min⁻¹ and immobilized on the sensor chip via the anti-GST antibody. Forty microliter of His-FlgN of various concentrations in the binding buffer to monitor association was passed over the sensor surface and then washed with the buffer to monitor dissociation at a flow rate of 20 µl min⁻¹. An acidic buffer (10 mM Glycine-HCl, pH 2.2) was used for regeneration of the surface of the sensor chip by removal of the captured proteins and any associates. All experiments were done at 25 °C. To obtain the K_D value, we analyzed SPR profiles using BIAevaluation software version 4.1 as described in the manufacturer’s instructions (GE Healthcare). At least three independent SPR measurements were carried out.

Surface plasmon resonance (SPR) analyses were carried out at 25°C using a Biacore 3000 instrument (GE Healthcare) with HBS-EP (10 mM sodium Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20) as running buffer. Anti-GST antibody (GST Capture Kit, GE Healthcare) was immobilized by amine coupling to a level of 9210 response units (RU) on to a CM5 sensor chip (GE Healthcare) according to the manufacturer's instructions. GST–hArc fusion protein (3 μg/ml, 66 nM) was reversibly captured at a flow rate of 30 μl/min and a 3-min injection to a level of 280–320 RU. Interaction with PS1 was tested by injecting a series of dilutions of the peptide (100–400 μM) over the GST–hArc surfaces at a flow rate of 30 μl/min and a 2-min injection. Peptide A (EQLTKCEVFRELKDLKGY; obtained from CPC Scientific Inc.), corresponding to the N-terminal region of α-lactalbumin, was treated in the same way as PS1 and used as a negative control. Regeneration of anti-GST antibody surfaces was accomplished with a 30 μl/min injection of 10 mM glycine/HCl, pH 2.1, for 2 min. All data generated were subtracted from the reference surface (no anti-GST antibody immobilized). BIAevaluation software (Version 3.2; GE Healthcare) was used for analysis of the sensorgrams. Dissociation constants (K_d) were calculated by fitting the sensorgrams to a 1:1 Langmuir binding model via non-linear regression analysis.

SPR measurements were performed on a Biacore 2000 instrument and were performed at room temperature in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20). GST-fusion proteins were captured to a CM5 Sensor Chip (GE Healthcare Life Sciences) by using a GST capture kit (GE Healthcare Life Sciences) according to the manufacturer’s instructions. Purified bovine ubiquitin was from Millipore-Sigma (U6253).

SPR experiments were performed using a Biacore T200 (GE Healthcare, Chicago, IL, USA). Approximately 8000 response units (RU) of the anti-GST antibody were immobilized on a Series S Sensor Chip CM5 (GE Healthcare) using a standard amine coupling protocol with a GST Capture Kit (GE Healthcare). Typically, 800–1000 RU of GST-Vpr were captured on the chip for each run. A buffer solution (10 mM HEPES pH 7.4, 150 mM NaCl, and 0.005% TWEEN20) containing 5% DMSO was used as the running buffer. Analyte solutions were prepared by adding a DMSO solution of compounds to the buffer to adjust the DMSO concentration to 5%. A solvent correction was performed using a buffer containing 4–6% DMSO.
The binding analysis was conducted at a flow rate of 30 μL/min at 25 °C. In each run, the association phase (60 s) and subsequent dissociation phase (60 s) were monitored. The dissociation constant (Kd) of the analyte was determined from the reference-subtracted sensorgrams by global fitting to a simple 1:1 binding model.