Cells were lysed with high-efficiency RIPA buffer (Solarbio) containing protease and phosphatase inhibitors, or with Nuclear Protein Extraction kit (BestBio, Shanghai, China) to obtain total protein or nuclear protein. Protein concentration was determined using a BCA Assay kit (Genstar, Beijing, China). Equal amounts of proteins were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Millipore, Billerica, USA). The membranes were blocked with 3% BSA, and incubated with specific primary antibodies overnight at 4°C, followed by incubation with the corresponding HRP-conjugated secondary antibody (Proteintech, Chicago, USA). Finally, the protein bands were visualized using the Chemiluminescence kit (Biotanon, Shanghai, China), and quantified using Gel-Pro analyzer (Media Cybernetics, Maryland, USA). GAPDH is used as the internal control. The following primary antibodies were used: anti-GAPDH antibody (Cell Signaling Technology, Beverly, USA), anti-Histone H3 antibody (Abcam), anti-GPER1 antibody (Abcam), anti-ERK1/2 antibody (Cell Signaling Technology), anti-phospho-ERK1/2 antibody (Cell Signaling Technology), and anti-phospho-NF-κB antibody (Cell Signaling Technology).
High efficiency ripa buffer
Manufactured by Solarbio
Sourced in China
High-efficiency RIPA buffer is a lysis buffer used for the extraction and purification of proteins from cells and tissues. It is designed to efficiently solubilize a wide range of proteins, including those that are membrane-bound or difficult to extract. The buffer contains a combination of detergents, salts, and other components that help to maintain the native structure and function of the extracted proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Anti erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti histone h3 antibody, by Abcam (1 mentions)
Nuclear protein extraction kit, by BestBio (1 mentions)
Anti phospho erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho nf κb antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Gel pro analyzer, by Media Cybernetics (1 mentions)
Primary antibody against vegf, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Nonfat milk, by BD (1 mentions)
2 protocols using high efficiency ripa buffer
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of VEGF
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TEC adipose tissue was homogenized and lysed using high-efficiency RIPA buffer (Solarbio, Beijing, China) or an NE-PER nuclear and cytoplasmic extraction kit (Boster, Pleasanton, CA, USA) with protease and phosphatase inhibitors (Roche, Mannheim, Germany). Equal quantities of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) followed by transfer to PVDF membranes (Millipore, Burlington, MA, USA). The blots were blocked with 5% nonfat milk (BD, Franklin Lakes, NJ, USA) or 5% BSA (Amresco, Houston, TE, USA) dissolved in 1× TBST (blocking buffer). The blots were then probed with a primary antibody against VEGF (1:2000; Abcam, Cambridge, MA, UK) dissolved in blocking buffer, followed by alkaline phosphatase-conjugated mouse IgG (1:5000; Abcam, Cambridge, MA, UK) as the secondary antibody. HRP (Millipore, Burlington, Massachusetts, USA) chemiluminescence signals were detected using a chemiluminescence imaging analysis system (Tanon, Shanghai, China). Densitometry of the immunoblot images was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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