Goat anti mouse igg conjugated to horseradish peroxidase

Ad-MSCs and BMP-7-MSCs were used for Western blot analysis. Briefly, the cells were washed twice with DPBS, and sonicated in lysis buffer (150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris at pH 7.5, 2 mM ethylenediaminetetraacetic acid) on ice for 30 min. Lysates were cleared by centrifugation (10 min at 13,000× g and 4 °C), and protein concentrations were determined using the Bradford method [37 (link)]. Equal amounts of protein (15 μg) were resolved by electrophoresis on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membrane blots were washed with TBST (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween-20), blocked with 5% skim milk for 1 h, and incubated with the appropriate primary antibodies at the recommended dilutions. The antibodies used included antibodies against actin (A3853, Sigma-Aldrich), BMP-7 (ab56023, Abcam, Cambridge, UK). The primary antibodies (1:1000) were diluted in TBST. The membrane was then washed, and primary antibodies were detected with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (1:5000, Invitrogen, Carlsbad, CA, USA). Bands were visualized using enhanced chemiluminescence (Invitrogen).

After SDS-PAGE separation onto 4-12% PAGE (150 V for 1.5 h) and electroblotting (30 V for 1.5 h) (Invitrogen, Life Technologies, Darmstadt, Germany) onto Amersham hybond-ECL nitrocellulose membrane (0.45 µm) (GE Healthcare GmbH, Little Chalfont, UK) non-specific binding of proteins to the membrane was blocked by incubation in PBS containing 3% BSA (Sigma-Aldrich, Munich, Germany) at room-temperature.
The membrane was then probed with primary antibodies either anti-ALADIN (mouse, B-11: sc-374073; 1:100 in 3% PBS/BSA) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), anti-PGRMC2 (rabbit, HPA041172; 1:200 in 5% PBS/milk powder) (Sigma-Aldrich, Munich, Germany) or anti-PGRMC2 (mouse, F-3: sc-374624; 1:100 in 3% PBS/BSA) (Santa Cruz Biotechnology, Inc.) overnight at 4°C. Secondary antibodies goat anti-mouse IgG conjugated to horseradish peroxidase (1:2000 in 3% PBS/BSA) (Invitrogen, Life Technologies, Darmstadt, Germany) or goat anti-rabbit IgG conjugated to horseradish peroxidase (1:3000 in 5% PBS/milk powder) (Cell Signalling Technology Europe B.V., Leiden, Netherlands) were incubated for 1 h at room-temperature.

Antibodies specific to antigens in different A. baumannii strains were measured by whole cell ELISA. Briefly, A. baumannii were grown to late log-phase (OD_600nm = 0.8, approximately 10⁹ CFU/ml), washed, and resuspended in PBS. 96-well plates (Brand plates, IMMUNOGrade) were coated with 50 µl of this bacterial suspension, overnight at 4°C, inactivated using 50 µl of 4% paraformaldehyde for 10 minutes, then blocked with 5% non-fat milk/0.05% Tween 20/PBS (blocking buffer) for 3 hours at room temperature. Sera were diluted to a 1:100 dilution in blocking buffer before addition, and binding to bacterial antigens detected with goat anti-mouse IgG conjugated to Horse-radish peroxidase (Invitrogen) and detected using the TMB substrate.