Total protein was purified from cells by RIPA lysis (keygen Biotech, China). Protein concentration was quantified by Bradford Protein Assay Kit (Beyotime). Equal amounts of protein were separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Subsequently, PVDF membrane was blocked by 5% bovine serum albumin for 2 h at room temperature prior to incubating overnight at 4 °C in respective primary antibodies. Membranes were then incubated for 2 h at room temperature with the appropriate secondary antibodies. The bands were exposed by applying chemiluminescence kit (Beyotime) in an imaging system (Bio-Rad, USA). Quantification of band intensity was also performed by ImageJ (National Institutes of Health, USA).
Ripa lysis
Manufactured by Keygen Biotech
Sourced in China
RIPA lysis is a buffer used to extract and solubilize proteins from cells and tissues. It contains a combination of detergents, salts, and other components that disrupt cell membranes and protein-protein interactions, allowing for the extraction of a wide range of proteins for further analysis.
Automatically generated - may contain errors
2 protocols using ripa lysis
1
Protein Purification and Quantification
2
Western Blot Analysis of Protein Expression
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Proteins were extracted with RIPA lysis (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) for 30 min at 4°C and measured by BCA protein assay. Protein samples (40 µg) were separated by 10% SDS-PAGE (Suzhou Well-Bridge Biological Technology Co., Ltd, Suzhou, China) and transferred to PVDF membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Following blocking with 5% non-fat milk in PBST for 2 h at 37°C, membranes were incubated overnight with primary antibodies at 4°C. Following washing membranes with PBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000) (cat. no. SA00001-2) and HRP-conjugated goat anti-mouse IgG (1:10,000) (cat. no. SA00001-1) (both from SanYing Company, Wuhan, China) for 120 min at room temperature. The bound antibody was detected by HRP substrate. All samples were performed in triplicate. The details of the primary antibodies used were as follows: Anti-human antigen R (HuR; cat. no. ab136542), anti-PLAGL2 (cat. no. ab139509) (Abcam, Cambridge, UK), anti-C-MYC (cat. no. 10828-1-AP) and anti-CD44 (cat. no. 15675-1-AP) (both from ProteinTech Group, Inc., Chicago, IL, USA) at a 1:1,000 dilution; and anti-GAPDH (cat. no. ab181602; Abcam) at a 1:3,000 dilution.
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