Rabbit igg antibodies

250nm SPIO carboxyl functionalised magnetic nanoparticles (Micromod) were covalently coated with anti-Frizzled 2 (Abcam), Trek1 (Alomone labs), Rabbit-IgG antibodies (Abcam) or RGD tri-peptide (Sigma) by carbodiimide activation as described previously [33 (link)]. Briefly, particles were activated using EDAC and NHS dissolved in 0.5M MES buffer pH6.3 (Sigma) for 60 mins. at room temperature with constant mixing. The particle suspension was washed and re-suspended in 0.1M MES buffer containing 40μg of anti-rabbit secondary antibody (Abcam) or 50μg RGD. The particle suspension was continuously mixed overnight at 4°C then washed and re-suspended in 0.1mL MES buffer containing 10μg of either anti-Frizzled 2 antibody (Abcam), anti-Rabbit-IgG (Abcam) or Anti-Trek1 antibody (Alomone labs). Particle suspensions were mixed for a further 3h at room temperature then blocked with 25mM Glycine (Sigma) for 30mins before final washing and re-suspension in 0.1% BSA in PBS (Fisher). Functionalised nanoparticles were then analysed for surface charge and size using a Zetasizer 3000 HSa (Malvern Instruments). Particles were diluted in dH₂0 and measurements performed at 25°C. The size and surface charge of coated nanoparticles was compared to uncoated activated nanoparticles.

After treatment, cells were harvested and lysed, and protein concentrations were measured by using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA, USA). All samples were separated on SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking the membranes with 5% (w/v) non-fat dry milk in TBS containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature, they were immunoblotted with the following monoclonal primary antibodies: vimentin, E-cadherin, MMP-2, MMP-9, phosphor-ERK1/2, total-ERK1/2, NF-kB subunit p65, β-actin, and GAPDH (all from Cell Signaling Technology, Beverly, MA,USA). Appropriate horseradish peroxidase-conjugated secondary antibodies, including mouse IgG and rabbit IgG antibodies (Abcam, Cambridge, MA, USA), were applied and incubated for 1 h at room temperature. Specific signals were visualized using a chemiluminescence (ECL) detection kit (Millipore).