The protein of exosomes extracted from 200 µl of prostatic fluid and cells in 1 well of a 6-well plate was extracted using RIPA Lysis Buffer (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Protein concentrations were determined using the BCA protein assay kit (Beyotime Institute of Biotechnology). Protein samples (20 µg) in each group were resolved on 8% gels using SDS-PAGE and transferred to polyvinylidene fluoride membranes. After being blocked using 5% non-fat milk for 1 h at room temperature, membranes were incubated overnight at 4°C with the following primary antibodies: Anti-CD63 (1:2,000; WL02549; Wanleibio, Co., Ltd.), anti-tumor susceptibility gene 101 (TSG101; 1:1,000; WL05130; Wanleibio Co., Ltd.), anti-RB1 (1:1,000; WL02216; Wanleibio, Co., Ltd.) and anti-GAPDH (1:1,000; WL01114; Wanleibio, Co., Ltd.). Following washing of the membranes three times with TBS-Tween-20 (0.05% Tween), the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG H+L secondary antibodies (1:10,000; WLA023a; Wanleibio, Co., Ltd.) for 2 h at room temperature, and the visualized using ECL chemiluminescence reagent (Beyotime Institute of Biotechnology). The protein expression levels were semi-quantified using ImageJ software (version, 1.52a; National Institutes of Health).
Wl02549
Manufactured by Wanlei
Sourced in China
The WL02549 is a laboratory equipment product. It serves as a core function to support laboratory operations.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Ecl chemiluminescence reagent, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab155282, by Abcam (1 mentions)
Gtx70255, by GeneTex (1 mentions)
Rs23910, by Immunoway (1 mentions)
Ac021, by ABclonal (1 mentions)
A13309, by ABclonal (1 mentions)
Rs23920, by Immunoway (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ac002, by ABclonal (1 mentions)
2 protocols using wl02549
1
Exosomal Protein Extraction and Quantification
2
Protein Expression Analysis via Western Blot
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The total proteins were extracted from cells or tissues using 1 × RIPA lysis buffer supplemented with a protease inhibitor cocktail (Roche). The concentrations of extracted total protein samples were measured by a BCA Protein Assay Kit (Beyotime). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membranes. After blocking the membranes with non-fat milk (5% w/v) for 1 h, the proteins were incubated at 4 °C overnight with the primary antibodies against CD63 (1:1,000, WL02549, Wanleibio, China), CD81 (1:1,500, GTX101768, Gene Tex, USA), TSG101 (1:1,500, GTX70255, Gene Tex), ACSL4 (1:10,000, ab155282, Abcam, UK), GPX4 (1:1,000, A13309, Abclonal, USA), GAPDH (1:5,000, AC002, Abclonal), or β-Tubulin (1:5,000, AC021, Abclonal). Next, the membranes were incubated with secondary anti-mouse or anti-rabbit antibodies (RS23910 and RS23920, ImmunoWay, USA) in dark at room temperature for 1 h. The membranes were scanned, and the gray values of protein band were detected by Odessey CLx (LI-COR, USA).
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