Pierce bca kit protocol

All cells were grown to 80% confluence. The media was then change to media without FBS (ovarian cancer carcinoma cell lines) or Ultroser G serum substitute (Pall Life Sciences) (FT cell lines) and the cells were cultured for an additional 72 h. The conditioned medium was then cleared by centrifugation and concentrated using a Millipore Amicon Ultra-15 centrifugal filter (Millipore Sigma). Protein content of conditioned medium was quantified using the Pierce BCA kit protocol (Thermo Fisher Scientific) and western bot was performed as described below.

Samples were lysed using RIPA buffer supplemented with protease and phosphatase inhibitor (Thermo Fisher Scientific). Samples were sonicated as described (44 (link)). Protein content of whole-cell lysates was quantified using Pierce BCA kit protocol (Thermo Fisher Scientific). Proteins were separated on a 4%–15% gradient SDS-PAGE (Bio-Rad) before being transferred to a PVDF membrane using the Turbo Blot system (Bio-Rad). Membranes were incubated with primary antibody overnight at 4°C (Supplemental Table 3). After washing, membranes were incubated with an anti-rabbit HRP-conjugated secondary antibody for 1 hour. Proteins were detected using Pico PLUS Chemiluminescent substrate (Thermo Fisher Scientific) and visualized with a ChemiDoc imaging system (Bio-Rad).

Whole cell lysates were prepared using M-PER buffer (Thermo Fisher Scientific). Protein content of whole cell lysate was quantified using the Pierce BCA kit protocol (Thermo Fisher Scientific). Proteins (20–30 µg) were separated on a 4–20% gradient SDS-PAGE before being transferred to a PVDF membrane using the Turbo Blot system (Bio-Rad). Membranes were incubated with primary antibodies overnight at 4 °C (see Supplementary Table 3). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins were detected using Clarity Chemiluminescent HRP Antibody Detection Reagent (Bio-Rad) and visualized with a Chemi-Doc imaging system (Bio-Rad). The uncropped blots for all the figures can be found in Supplementary Figs. 1–4.