Mouse monoclonal antibody for β actin

Rabbit monoclonal antibodies for Vimentin, E-Cadherin, N-Cadherin, Zeb-1 (as a part of the EMT antibody sampler kit), anti-Rabbit IgG and anti-mouse IgG HRP linked secondary antibodies were obtained from Cell Signaling (Danvers, MA). Mouse monoclonal antibody for β-Actin was purchased from Sigma Aldrich and for active-β-Catenin was obtained from EMD Millipore (Billerica, MA). All the primary antibodies were diluted according to the manufacturer’s instruction in TBST with 1% BSA. Secondary antibodies were prepared at a dilution of 1:1000 in TBST with 1% blocking grade milk from Bio-Rad (Hercules, CA). Antibody against E-Cadherin conjugated with Alexa-Fluor 647 for flow cytometry was purchased from BD Biosciences (Franklin Lakes, NJ).

Briefly, equal amounts of total cellular proteins (20 μg) were resolved on 10% SDS-PAGE gel and electro-transferred onto a nitrocellulose membrane. Western blot analysis was conducted as described previously [30 (link)]. Rabbit polyclonal antibodies for phosphorylated-LRP6 and non-phosphorylated-β-catenin were purchased from Cell Signaling (Danvers, MA); a rabbit polyclonal antibody for VEGF-A was purchased from Santa Cruz (Dallas, TX); a mouse monoclonal antibody for β-actin was purchased from Sigma Aldrich (Saint Louis, IL) and an anti-Fzd7 mouse monoclonal antibody was purchased from LifeSpan Biosciences (Seattle, WA).

Approximately 30–40 μg of protein from each sample was mixed with SDS loading buffer (Lamelli sample buffer (2X) containing 2-Mercaptoethanol). The proteins were separated by Novex 4–20% Tris-Glycine gel and then transferred onto a nitrocellulose membrane (Novex, Life Technologies). The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween-20 at room temperature for an hour. The membrane was washed four times and incubated overnight at 4°C with either mouse monoclonal antibody to P-gp (C219; Pierce, Thermo Scientific) at 1:1000 dilution or a rabbit monoclonal antibody for HER-2/ErbB2 (Cell Signaling Tech., Danvers, MA) at a dilution of 1:1000. The blot was normalized by incubating with mouse monoclonal antibody for β-actin (Sigma-Aldrich) at 1:20,000 dilution. After the primary antibody incubation, the membrane was washed four times and then incubated with the secondary antibody conjugated with horse-radish peroxidase (anti-mouse or anti-rabbit) (GE HealthCare) at a dilution of 1:20,000 for 1 hour. The protein expression was detected using the ECL reagent (GE Healthcare) on a high performance chemiluminescence film (Thermo Scientific).