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Preprint

Western blot assay

CM Chenkui Miao
LJ Li Jia

Last updated date: May 30, 2022 Views: 639 Forks: 0

original An abbreviated version of this protocol was published in Science Advances in Feb, 2022
RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer
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As you can see, the Western Blot protocol is included in the method part, see below in detail:

 

PCa cells were treated as indicated and harvested for protein extraction. Cells were rinsed with phosphate-buffered saline (PBS),
scraped, and lysed in cold radioimmunoprecipitation assay (RIPA) Lysis and Extraction Buffer (#89900, Thermo Fisher Scientific) containing Protease and Phosphatase Inhibitor Cocktail (#78447, Thermo Fisher Scientific). Protein concentration was quantified using the Pierce BCA Protein Assay Kit (#23225, Thermo Fisher Scientific) and measured with a spectrophotometer. Western blot was performed as previously described (58) and repeated at least two times. Molecular weight markers were used to determine the size of proteins. Protein bands were quantified using ImageJ software. 

 

Antibodies were listed in table S3. You may check it. 

 

Best,
 

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Miao, C and Jia, L(2022). Western blot assay. Bio-protocol Preprint. bio-protocol.org/prep1691.
  2. Miao, C., Tsujino, T., Takai, T., Gui, F., Tsutsumi, T., Sztupinszki, Z., Wang, Z., Azuma, H., Szallasi, Z., Mouw, K. W., Zou, L., Kibel, A. S. and Jia, L.(2022). RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer. Science Advances 8(7). DOI: 10.1126/sciadv.abl9794

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