Secondary anti rabbit igg antibody conjugated to horseradish peroxidase

To analyze the expression levels of the µ-opioid receptor OPRM1 under treatment with doxorubicin, carboplatin, and vincristine, whole cell extracts from RD, RH30, and SkMC were prepared using RIPA buffer (Cell Signaling Technology, Inc.) and incubated on ice for 30 min. The whole cell extracts were centrifuged at 4 °C, 14,000×g for 20 min, and the protein concentration of the supernatant was determined by Bradford assay (Bio-Rad laboratories GmbH, Feldkirchen, Germany). Lysates (30 µg) were subjected to 10% SDS-PAGE, and proteins transferred to a nitrocellulose membrane (VWR International GmbH), and the membranes were blocked for 1 h at room temperature with 10% non-fat dried milk (Carl Roth GmbH & Co. KG) in TBS-T. For immunoblotting, the membranes were incubated over night at 4 ºC with an antibody directed against OPRM1 (1:200; Thermo Scientific Technology, #PA5-26138). Anti-GAPDH antibody (1∶1,000; Cell Signaling Technology, Inc., #2118S) was used as a loading control. After incubation for 1 h at room temperature with a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (1∶3,000; Cell signaling Technology, Inc., #7074S), the proteins were visualized by the WesternSure^® PREMIUM Chemiluminescent Substrate (LI-COR Biosciences). Specific bands were quantified with the Odyssey Fc Imaging System (LI-COR Biosciences).

To analyze the expression levels of PDE5, whole-cell extracts were prepared using RIPA buffer (Cell Signaling Technology, Inc.) incubated on ice for 30 min. The whole-cell extracts were centrifuged at 4 °C, 14,000×g for 20 min, and the protein concentration of the supernatant was determined by Bradford assay (Bio-Rad labraratories GmbH). Lysates (30 µg) were subjected to 10% SDS-PAGE, and proteins transferred to a nitrocellulose membrane (VWR International GmbH). Membranes were blocked for 1 h at room temperature (RT) with 10% non-fat dried milk (Carl Roth GmbH & Co. KG) in TBS (Sigma-Aldrich; Merck KGaA) containing 0.1% Tween-20 (Carl Roth GmbH & Co. KG). For immunoblotting, the membranes were incubated overnight at 4 °C with an antibody directed against PDE5 (1:1,000; Cell Signaling Technology, Inc., #2395S). Anti-GAPDH antibody (1∶1,000; Cell Signaling Technology, Inc., #2118S) was used as a loading control. After incubation for 1 h at RT with a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (1∶3,000; Cell Signaling Technology, Inc., #7074S), the proteins were visualized by the WesternSure^® PREMIUM Chemiluminescent Substrate (LI-COR Biosciences). Specific bands were quantified with the Odyssey Fc Imaging System (LI-COR Biosciences) (Schmid et al. 2014 (link)).