Total protein was extracted from cells using enhanced radioimmunoprecipitation assay lysis buffer (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) containing protease inhibitor, and the concentration was determined using a bicinchoninic acid protein assay (Boster). The protein was then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto polyvinylidene fluoride membranes. The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and probed at 4°C overnight with the following diluted primary antibodies (Abcam): HO-1 (ab68477, 1:10,000, Rabbit), Occludin (ab216327, 1:1,000, Rabbit), SP1 (ab227383, 1:2,000, Rabbit), HDAC4 (ab235583, 1:1,000, Rabbit), HMGB1 (ab18256, 1:500, Rabbit), Acetyl-Lys (ab190479, 1:2,000, Rabbit), GAPDH (ab181603, 1:5,000, Rabbit), and Lamin A (ab133256, 1:10,000, Rabbit). After 3 washes using Tris-buffered saline Tween-20, the membrane was reprobed with the horseradish peroxidase-labeled secondary antibody (goat anti-rabbit, ab205718, 1:1,000; goat anti-mouse, ab205719, 1:1,000; Abcam) for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence (Baoman Biotechnology Co., Ltd., Shanghai, China). With GAPDH and Lamin A as internal references, ImageJ software was used to analyze the gray value.
Enhanced radioimmunoprecipitation assay lysis buffer
Manufactured by Boster Bio
Sourced in China
Enhanced radioimmunoprecipitation assay lysis buffer is a reagent used to extract and solubilize cellular proteins for analysis. It is designed to disrupt cell membranes and release proteins while maintaining protein-protein interactions.
Automatically generated - may contain errors
Lab products found in correlation
Ab227383, by Abcam (1 mentions)
Ab181603, by Abcam (1 mentions)
Ab133256, by Abcam (1 mentions)
Bicinchoninic acid protein assay, by Boster Bio (1 mentions)
Ab190479, by Abcam (1 mentions)
Ab68477, by Abcam (1 mentions)
Ab216327, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab18256, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Ab6808, by Abcam (1 mentions)
Bicinchoninic acid kit, by Boster Bio (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Lin28b, by Abcam (1 mentions)
Immobilon p, by Merck Group (1 mentions)
As014, by ABclonal (1 mentions)
Ac033, by ABclonal (1 mentions)
Chemidoc xrs plus luminescent image analyzer, by Bio-Rad (1 mentions)
Gapdh, by ABclonal (1 mentions)
4 protocols using enhanced radioimmunoprecipitation assay lysis buffer
1
Protein Expression Analysis by Western Blot
2
Protein Extraction and Western Blot Analysis
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The total protein was extracted from cells or the EVs with enhanced radioimmunoprecipitation assay lysis buffer (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) with protease inhibitor. After electrophoresis-separation and electro-transfer, the polyvinylidene fluoride membranes were then blocked using 5% bovine serum albumin (BSA) at room temperature for 2 h and underwent overnight incubation at 4 °C with the diluted primary antibodies against GPR158 (ab121388, 1:1000), CD9 (ab223052, 1:1000), CD81 (ab109201, 1:1000), Alix (ab88388, 1:1000), Calnexin (ab22595, 1:1000), and GAPDH (ab8245, 1:5000) as well as with horseradish peroxidase (HRP)-labeled secondary goat anti-nude mouse antibody (ab6808, 1:2000, Abcam). The immunocomplexes on the membrane were visualized using enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA) and band intensities were quantified using ImageJ software, with GAPDH serving as a loading control.
3
Western Blot Analysis of Protein Expression
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The cultured cells were collected and lysed with enhanced radioimmunoprecipitation assay lysis buffer (Boster Biological Technology, Wuhan, P.R. China) containing protease inhibitors. The bicinchoninic acid kit (Boster Biological Technology) was utilized to determine the protein concentration. Proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the separated proteins were electro-transferred to polyvinylidene fluoride membrane (Immobilon P, Millipore, Billerica, MA, USA), and the membrane was treated with 5% skimmed milk at ambient temperature for 2 h to block non-specific binding. The membrane was incubated with primary antibody at 4°C overnight and then with horseradish peroxidase conjugated secondary antibody at 37°C for 1 h. Enhanced chemiluminescence reagent (Thermo Fisher Scientific) was utilized to visualize the immunoreactive bands, followed by imaging using ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA). ImageJ analysis software was utilized to quantify the gray value of protein bands. Antibodies included SIRT6 (A7416, 1:2,000, ABclonal, Woburn, MA, USA), NRP-1 (A19087, 1:2,000, ABclonal), GAPDH (AC033, 1:50,000, ABclonal), Lin28B (ab191881, 1:2,000, abcam), rabbit secondary antibody (AS014, 1:10,000, ABclonal), and murine secondary antibody (AS003, 1:10,000, ABclonal).
4
Western Blot Analysis of Epigenetic Regulators
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The cells collected after trypsin treatment were lysed with enhanced radio-immunoprecipitation assay lysis buffer (Boster, Wuhan, China). Following separation with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein was electro-blotted to a polyvinylidene fluoride membrane. After undergoing a 2 h membrane blocking with 5% bovine serum albumin (BSA) at ambient temperature, membrane incubation was carried out overnight with the diluted primary rabbit antibodies (1:1000, Abcam) against EZH2 (ab186006), TET1 (ab191698), cyclin A (ab181591), and p53 (ab131442) at 4 °C. The membrane was re-probed with HRP-tagged goat anti-rabbit secondary antibody (1:2000, Abcam, ab205719) at ambient temperature for 1 h before development with enhanced chemiluminescence (Millipore, Billerica, MA). The gray value of bands in western blot images was quantified utilizing the Image J software, with β-actin (ab8226, 1:1000, Abcam, mouse) as a normalizer. Each experiment was repeated 3 times.
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