Surface plasmon resonance saturation experiments were performed on a Biacore 8K instrument (GE Healthcare) at 25°C using a Series S Sensor Chip NTA (GE Healthcare) and SPR running buffer (10 mM HEPES, 150 mM NaCl, 50 μM EDTA, 0.05% v/v Tween 20, pH 7.4). Solutions of His6-tagged CSP27 and CSP9 were prepared in SPR running buffer at concentrations of 0.1 μg/ml and 0.4 μg/ml, respectively. His6-tagged CSP27 and CSP9 were immobilized on separate channels on the sensor chip surface as per the manufacturer’s recommendations: a pre-conditioned chip was first activated with 500 μM NiCl2, and a solution of the His6-tagged ligand was subsequently passed over the chip using a flow rate of 5 μl/min for 120 s. This yielded approximately 150 RU and 50 RU of immobilized CSP27 and CSP9, respectively. A saturating solution of mAb 2A10 (2 μM in SPR running buffer) was then passed over the chip for 400 s using a flow rate of 30 μl/min, followed by a 400 s dissociation period. The increase in response units (RU) corresponding to ligand immobilization (RUlig) and analyte binding (RUanalyte) in the reference-subtracted (reference = blank surface) sensorgrams was measured, and the binding stoichiometry (n, molar ratio of antibody to antigen in the complex under saturating concentrations of mAb 2A10) was estimated using Equation (1) as previously described (Oda and Azuma, 2000), using molecular weights (MW) calculated using ProtParam (Wilkins et al., 1999):CSP27 (35.4 kDa), CSP9 (28.2 kDa) and mAb 2A10 (145.9 kDa).

All buffers were filtered and degassed prior to use. Following each cycle, the chip was completely regenerated using sequential washes of 500 mM imidazole, 350 mM EDTA (pH 8.5) and 100 mM NaOH. Each experiment was performed in duplicate (n = 2), on separate channels on the SPR chip.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.