The cells were seeded in six-well plates at a density of 5 × 104 cells/mL per well and treated with acacetin (0, 20, 40, and 60 μM) for 24 h. After washing the cells with PBS, the cells were lysed in ice-cold RIPA for 30 min. The lysis buffer contained a mixture of protease and phosphatase inhibitors. Subsequently, the cells were centrifuged at 12,000 ×g for 15 min at 4 °C, and the supernatant was collected. A BSA Protein Assay (Beyotime, Shanghai, China) was used to quantify the protein concentration according to the manufacturer’s instructions. The proteins were separated using SDS-PAGE gels (8–12% Tris-SDS gels) at 110 V for 0.5–1.5 h and wet-transferred to PVDF membranes at 300 mA. After blocking the membranes with 5% nonfat milk for an hour at room temperature, the membranes were incubated with the primary antibody at 4 °C overnight. The membranes were washed three times with TBST buffer and then probed with an anti-rabbit (1:5000) or anti-mouse (1: 5000) secondary antibody tagged with horseradish peroxidase for 1 h at room temperature. The bound immunocomplexes were detected using ChemiDoc XRS (BioRad).
Bsa protein assay
Manufactured by Beyotime
Sourced in China
The BSA Protein Assay is a colorimetric-based method used to quantify the total protein concentration in a sample. It utilizes the reaction between copper ions and peptide bonds to produce a colored complex, which can be measured spectrophotometrically. The assay is designed to provide a simple and reliable way to determine protein levels, making it a useful tool for various applications in research and analytical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using bsa protein assay
1
Acacetin-Mediated Protein Expression Analysis
2
Western Blot Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell proteins were promptly homogenized in a homogenization buffer containing 1 M TrisHCl pH 7.5, 1% Triton X-100, 1% Nonidet P-40 (NP-40), 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, and 1 mM PMSF, and centrifuged at 10, 000×g for 30 min to collect the supernatant. Protein concentrations were established using a BSA protein assay (Beyotime, SH, China). The total cellular protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Roche, Basel, Switzerland). After the membranes were blocked in 5% nonfat milk in TBST (150 mM NaCl, 20 mM Tris, 0.05% Tween 20) for 2 h, they were incubated with their primary antibodies overnight at 4°C. The membranes were washed with TBST three times for 5 min each, and the horseradish-peroxidase-linked IgG was used as the secondary antibody for 2 h at room temperature. The membrane was developed using the ECL detection systems. The experiments were performed on three separate occasions. The densitometry data were measured and analyzed using ImageJ software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!