Ovarian cancer cells were collected and lyzed in 50 mL cell lysis buffer containing protease inhibitors. The cell lysates were separated on 12% SDS–PAGE. After the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA), we blocked the proteins with Tris-buffered saline (TBS) and 0.1% Tween 20 (TBS/T) which containing 5% bovine serum albumin. The PVDF membranes were incubated with specific primary antibodies (Anti-UBE2N antibody: Abcam, 1:1000; Anti-Fos antibody: Cell Signaling Technology, 1:1000; Anti-P53 antibody: Cell Signaling Technology, 1:1000; Anti-GAPDH antibody: Cell Signaling Technology, 1:1000) at room temperature for 1 hour or at 4°C overnight. The membranes were washed three times with TBS/T and then incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. The protein concentration was quantified using the BCA Protein Kit (Applygen, Beijing, China). The protein GAPDH was used as an internal control. The relative expression of other proteins was normalized against GAPDH.
Bca protein kit
Manufactured by Applygen
Sourced in China
The BCA Protein Kit is a reagent-based assay for the quantitative determination of total protein concentration. It utilizes the bicinchoninic acid (BCA) method to measure the reduction of copper ions by proteins in an alkaline environment, resulting in a purple-colored complex that can be measured spectrophotometrically.
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2 protocols using bca protein kit
1
Western Blot Analysis of Ovarian Cancer Cells
2
Western Blot Analysis of RAS Proteins
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HCC cells and tissues from snap-frozen HepG2 xenografts were lysed using RIPA lysis buffer (Applygen Technologies, Beijing, China). Proteins were quantified using a BCA protein kit (Applygen). Proteins (50 μg) were separated by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk and incubated with primary antibodies. The membranes were washed in PBS-T (PBS and 0.1% Tween-20) and incubated with a peroxidase-conjugated secondary antibody (KPL, Gaithersburg, MD, USA), followed by development with a chemiluminescent substrate (Applygen). The Gel-Doc imaging system was used to scan images on Kodak film. Antibodies for KRAS, HRAS, and NRAS were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH and beta-actin (β-action) antibodies were obtained from Proteintech (Chicago, IL, USA).
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