Hrp goat anti rabbit igg secondary antibody

Prior to electrophoresis, samples were adjusted to 2% (w/v) SDS and 3 mM β-mercaptoethanol and boiled for 10 min. Proteins were resolved by SDS-PAGE with 10% (w/v) resolving gels and 5% (w/v) stacking gels, and PageRuler prestained molecular mass standards (Fermentas). Chemiluminescent western blot analysis was performed using the ECL system (Amersham Biosciences, Pittsburgh, PA, USA) as previously described [19 (link),32 (link)], with modification. Western blots were probed with 1:5000 dilution of the anti-amylase antibody [19 (link)], and then 1:5000 dilution of HRP goat anti-rabbit IgG secondary antibody (Zymed, Rockford, IL, USA). Purified amylase (40 ng) was used as a standard for Western blot analysis. Anti-amylase antibody was pre-adsorped using cell extracts from the amyA disruption strain, PBL2004 [19 (link)], to remove non-specific antibodies, as previously described [37 ] with modification. Pre-wetted nitrocellulose membrane (80 μg protein/cm²) was incubated with sonicated cell pellets for 3 h at room temperature, and blocked overnight in 2.5% (w/v) milk (Nestle, Glendale, CA, USA) in PBS buffer (10 mM NaH₂PO₄ and 150 mM NaCl). Nitrocellulose membranes were washed three times with PBS buffer and incubated with 1:5000 dilution of anti-amylase antibody in 0.5% (w/v) milk in PBS buffer for 2 h at room temperature.

All reagents and chemicals used in this study were purchased from Sigma (St. Louis, USA) unless otherwise specified. For cell culture, RPMI-1640 medium was purchased from PAA Laboratories GmbH (Austria). Tenascin C protein (Cat. No. CC065) was obtained from EMD Millipore (Billerica, Massachusetts, USA). Cilengitide was purchased from Selleckchem (Houston, USA). Rabbit anti-human integrin α_v and β₃ were from Cell Signaling Technologies, Inc. (Danvers, MA, USA). HRP goat anti-rabbit IgG secondary antibody was from Zymed (San Francisco, California, USA). Sequencing-grade trypsin was from Promega (Madison, MI, USA). Human Pan Monocyte Isolation Kits were purchase from Miltenyi Biotec GmbH (Germany) and 5-Carboxyfluorescein N-Succinimidyl ester (CFSE) fluorescent dye was purchase from Cayman Chemical (Michigan, USA).