Goat anti mouse igg polyclonal antibody

Protein extracts were prepared in lysis buffer (150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate, 1% Triton-X100, in 50 mM Tris–HCl pH 7.4), supplemented with a protease and phosphatase inhibitor cocktail (Biotool, Houston, TX, United States). Extracts were electrophoretically separated on 10% or 12% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, United States). The membranes were then blocked with 5% w/v BSA Fraction V in PBS containing 0.1% Tween-20, and subsequently incubated with anti-Phospho-Myosin Light Chain II (Ser19) (Cell Signaling Danvers, MA, United States), total Myosin Light Chain II (Cell Signaling Danvers, MA, United States), or anti-HSP90 (Santa Cruz Biotech) primary antibodies. The membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Labs, Inc., West Grove, PA, United States) or goat anti-mouse IgG polyclonal antibody (Bio-Rad Laboratories, Inc., Hercules, CA, United States) for 1 h at room temperature. Bands were visualized with a chemiluminescence kit (Pierce, Thermo Scientific, Rockford, IL, United States), using an enhanced ECL detection system (GE Healthcare).

Protein extracts were prepared in a lysis buffer (150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate, 1% Triton-X100, in 50 mM Tris-HCl pH 7.4) supplemented with a protease and phosphatase inhibitor cocktail (Biotool, Houston, TX, USA). Extracts were electrophoretically separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), which were blocked with 5% w/v nonfat, dry milk in PBS containing 0.1% Tween-20, and subsequently incubated with anti-β3 Integrin (Millipore, Billerica, MA, USA) or anti β-actin (Sigma-Aldrich) primary antibodies. The membrane was then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Labs, Inc., West Grove, PA, USA) or goat anti-mouse IgG polyclonal antibody (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 1 h at room temperature. Bands were visualized with a chemiluminescence kit (Pierce, Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions.

Recombinant proteins at a concentration of 1.5 mg/mL in 1 µL were adsorbed onto a nitrocellulose membrane. Next, the membranes were cultured in a blocking buffer containing PBS, 0.1% Tween-20 with 1% BSA. After washing three times with PBS + 0.1%, Tween-20 10H10 mAb 2 μg/mL in blocking buffer was added and incubated for 10 min. After incubation, membranes were washed three times and incubated for 10 min with goat anti-Mouse IgG polyclonal antibody 0.25 μg/mL conjugated with alkaline phosphatase (BioRad, Berkeley, CA, USA). After washing 3 times, membranes were incubated for 5 min with an aqueous solution of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitrosinetetrazole (NTB) (Invitrogen, Waltham, MA, USA). The enzymatic reaction was stopped by washing with distilled water.