The cells were washed three times with PBS, and radioimmunoprecipitation assay lysis buffer (Aspen Pharmacare Holdings Ltd.; cat. no. AS1004) was used to extract the total proteins from cells. Cell lysates (1 × 104) were subjected to 10% SDS-PAGE followed by determination of protein concentration by the bicinchoninic acid method. The proteins (50 μg) were then transferred onto a 10% SDS-PVDF membrane. The PVDF membrane was blocked by 5% bovine serum albumin (Abcam) at room temperature for 2 hours. A chemiluminescence detection system (Canon, Inc.; cat. no. LiDE110) was then used to visualize proteins based on the provided instructions. Antibodies used were as follows: anti-PTEN (1:1000; Abcam; cat. no. Ab32199), anti-Cyclin D1 (1:500; Abcam; cat. no. Ab134175), anti-Cyclin D3 (1:500; Abcam; cat. no. Ab112034), anti-Bcl-2 (1:500; Abcam; cat. no. Ab185002), anti-Bax (1:500; Abcam; cat. no. Ab182734), and anti-GAPDH (1:10,000; Abcam; cat no. ab37168). All experiments were conducted in triplicate.
Radioimmunoprecipitation assay lysis buffer
Manufactured by Aspen
Radioimmunoprecipitation assay (RIPA) lysis buffer is a solution used to extract and solubilize proteins from cells or tissues for various analytical techniques, such as Western blotting and immunoprecipitation. It contains a mixture of detergents, salts, and buffers that help to disrupt cell membranes and release the intracellular contents, including the proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (3 mentions)
Bovine serum albumin (bsa), by Abcam (2 mentions)
Anti bax, by Abcam (1 mentions)
Anti pten, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti cyclin d1, by Abcam (1 mentions)
Anti cyclin d3, by Abcam (1 mentions)
Chemiluminescence detection system, by Canon (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Anti cd9, by Abcam (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Anti osteocalcin ocn, by Abcam (1 mentions)
Runx2, by Abcam (1 mentions)
3 protocols using radioimmunoprecipitation assay lysis buffer
1
Protein Expression Analysis in Cells
2
Exosome Protein Profiling by Western Blot
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The cells were washed three times with PBS, and radioimmunoprecipitation assay lysis buffer (Aspen Pharmacare Holdings Ltd.; cat. no. AS1004) was used to extract the total proteins from the cells. The cell lysates (1x104) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the protein concentration was determined through the bicinchoninic acid method. The proteins (50 μg) were then transferred onto a 10% sodium dodecyl sulfate polyvinylidene difluoride membrane. The membrane was blocked with 5% bovine serum albumin (Abcam) at room temperature for 2 h. A chemiluminescence detection system (Canon, Inc.; cat. no. LiDE110) was then used to visualize the proteins based on the provided instructions. The following antibodies were used: anti-TSG101 (1:1000; Abcam, cat. no. Ab125011), anti-CD9 (1:1000; Abcam, cat. no. Ab92726), anti-NOX5 (OCN; 1:500; Abcam, cat. no. Ab198213) and anti-GAPDH (1:10,000; Abcam, cat no. Ab37168). All experiments were conducted in triplicate.
3
Osteogenic Protein Expression Analysis
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The cells were washed three times with PBS three times and radio immunoprecipitation assay lysis buffer (Aspen Pharmacare Holdings Ltd.; cat. no. AS1004) was used to extract the total proteins from cells. Cell lysates (1×104) were subjected to 10% SDS-PAGE followed by determination of protein concentration by the bicinchoninic acid method. The proteins (50 µg) were then transferred onto a 10% SDS-PVDF membrane. The PVDF membrane was blocked by 5% bovine serum albumin (Abcam) at room temperature for 2 h. A chemiluminescence detection system (Canon, Inc.; cat. no. LiDE110) was then used to visualize proteins based on the provided instructions. Antibodies used were as follows: Anti-collagen I (1:500; Abcam; cat. no. ab34710), anti- alkaline phosphatase (ALP; 1:1,000; Abcam; cat. no. ab95462), anti-Osteocalcin (OCN; 1:500; Abcam; cat. no. ab93876), anti-Runt-related transcription factor 2 (Runx2; 1:500; Abcam; cat. no. ab23981) and anti-GAPDH (1:10,000; Abcam; cat. no. ab37168). All experiments were conducted in triplicate.
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