Mes buffer ph6

Carboxyl functionalized magnetic nanoparticles (nano-mag®-D, 09-02-252, Micromod) were covalently coated with Anti-Activin Receptor type IIA (ActRIIA) antibody (ab135634) herein termed as "MNPs-ActRIIA", or with Anti-Rabbit-IgG Fc antibody (ab97196), "MNPs-IgG", by carbodiimide activation as described previously. 14 Briefly, particles were activated using EDAC (03449, Sigma) and NHS (130672, Sigma) dissolved in 0.5 M MES buffer pH6.3 (Sigma) for 1 h at room temperature under continuous mixing. The particle suspension was washed and re-suspended in 0.1 M MES buffer containing 60 μg of anti-rabbit secondary antibody (ab97196). The particle suspension was continuously mixed overnight at 4 °C and then washed and re-suspended in 0.1 mL MES buffer containing 10 μg of Anti-Activin Receptor type IIA antibody. Particle suspensions were mixed for 3h at room temperature and then blocked with 25mM Glycine (Sigma) for 30 minutes before final washing and re-suspension in distilled water. Functionalized nanoparticles were then analyzed for surface charge and size using a Zetasizer 3000 HSa (Malvern Instruments). Particles were diluted in dH 2 0 and measurements performed at 25 °C. The size and surface charge of ActRIIA-coated nanoparticles was compared to IgG-coated nanoparticles.