Proteins were lysed from cultured cells using RIPA Lysis and Extraction Buffer (G-Biosciences) with protease inhibitor cocktail (Roche), followed by incubation at 100°C for 20 min. Cell lysates were separated by electrophoresis on 4–20% gradient polyacrylamide gels and transferred to PVDF membranes. The blots were blocked with TBS-T (20 mM Tris–HCl, pH 7.6, 136 mM NaCl and 0.1% Tween-20) containing 5% BSA (bovine serum albumin) for 1 h and then incubated with primary antibody solution at 4°C overnight. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature (RT). Then the membrane was washed with TBS-T, followed by developing with ECL reagents (GE Healthcare). Antibodies used for western blotting were anti-Cdx2 (1:1000, ab88129, Abcam), anti-Arid3a (1:5000; (20 (link))), anti-Gata3 (1:1000, SC-9009, Santa Cruz), anti-β-actin (1:10000, ab20272, Abcam) and anti-Hdac1 (1:1000, ab7028, Abcam).
Ripa lysis and extraction buffer
Manufactured by G Biosciences
Sourced in Switzerland, United States
RIPA lysis and extraction buffer is a detergent-based buffer used for the lysis of cells and extraction of proteins. It contains a combination of ionic and non-ionic detergents that solubilize cellular membranes and proteins, allowing for the efficient extraction of proteins from cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Ab88129, by Abcam (1 mentions)
Ab7028, by Abcam (1 mentions)
Ab20272, by Abcam (1 mentions)
Sc 9009, by Santa Cruz Biotechnology (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Mircury rna isolation kit, by Qiagen (1 mentions)
Laemelli buffer, by Bio-Rad (1 mentions)
Mircurytmexosome isolation kit serum and plasma mircury, by Qiagen (1 mentions)
Irdye 800cw conjugated goat anti rabbit secondary antibody, by LI COR (1 mentions)
Bca protein assay kit, by G Biosciences (1 mentions)
Compartmental protein extraction kit, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
4 protocols using ripa lysis and extraction buffer
1
Protein Extraction and Western Blot Analysis
2
Exosome Isolation and Protein Analysis
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Pooled human serum (Valley Biomedical, Winchester, VA, catalog #HS1004P) and six commercially available individual donor samples (BioreclamationIVT, NY, Item #HMSRM) were used for exosome isolation. We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA). To isolate exRNA, miRCURY RNA Isolation Kit–Cell & Plant (Exiqon, Woburn, MA) was used. RIPA lysis and extraction buffer (GBiosciences, St. Louis, MO), 2xLaemelli buffer (Bio-Rad, Hercules, California), and 100x halt protease inhibitor EDTA-free cocktail (Thermo Scientific, Grand Island, NY) were used to prepare protein samples for western blot analysis. Rabbit monoclonal anti-CD63 antibody was from Abcam (Cambridge, MA), and rabbit polyclonal anti-CD9 antibody was purchased from Santa Cruz (Dallas, Texas). IRDye 800CW-conjugated goat anti-rabbit secondary antibody was from LI-COR (Lincoln, Nebraska).
3
Quantification and Characterization of Extracellular Vesicles
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The total protein content in isolated UEVs was quanti ed using a bicinchoninic (BCA) protein assay kit (G-Biosciences) following the manufacturer's instructions. Samples were stored at -80°C until further analysis. For SDS-PAGE, 10µg of UEV protein was lysed using RIPA lysis and extraction buffer (G-Biosciences) including protease inhibitor cocktail (Roche, Basel, Switzerland) followed by incubation at 4°C for 15min. The lysed protein samples were then mixed with reducing sample buffer and denatured for 10 mins at 70°C. The exosomal proteins were resolved on 10% SDS PAGE for 1.5 h at 120 V, and the gels were stained using 2% silver nitrate.
4
Protein Extraction and Analysis from Tumors
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Proteins were extracted from tumour tissues and matched normal livers with radioimmunoprecipitation assay (RIPA) Lysis and Extraction Buffer (G-Biosciences, St. Louis, MO, USA) supplemented with cOmplete Mini protease inhibitors (Roche, Mannheim, Germany) and phosphatase inhibitors (Roche). In Huh-28 and SNU-1079 cell lines, cytosolic and membrane protein fractions were extracted using the Compartmental Protein Extraction Kit (Millipore), as per the manufacturer's instructions. Equal amounts of protein extracts were then separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gels and transferred to polyvinylidene difluoride membranes by standard methods. Membranes were immunoblotted with the following primary antibodies: antieequilibrative nucleoside transporter (ENT-1) (Abcam), antiecaspase-3 (Cayman Chemical Company, Ann Arbor, MI, USA), antiepoly-(ADP-ribose)polymerase-1 (PARP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antiebactin (Cell Signalling, Danvers, MA, USA). Digital images of X-ray films were quantified by ChemiDoc XRSþ (Image Lab Software, Bio-Rad).
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