The radio-immunoprecipitation assay (RIPA; Rockland Immunochemicals, PA, USA) buffer was used to lyse the same numbers of EVs (1 × 1010 particles). The lysed EV solutions were placed onto the NC membrane of the Proteome Profiler™ Antibody Arrays Human XL Cytokine Array Kit (R&D Systems, MN, USA). After developing the NC membrane with ChemiDoc™ XRS+, the intensities of the cytokine array were quantified using ImageLab software. For data analysis, the average intensity of cytokine array was exported with expression of cytokine array intensities in logarithm base 2. The PANTHER (Protein Analysis THrough Evolutionary Relationships; http://www.pantherdb.org ) was utilized to analyze the representative proteins from the cytokine array. The gene ontology (GO) and KEGG pathway were analyzed using the DAVID (Database for annotation, visualization and integrated discovery; https://david.ncifcrf.gov/ ) for prediction of functionalities.
Radioimmunoprecipitation assay (ripa)
Manufactured by Rockland Immunochemicals
Sourced in United States
RIPA is a buffer solution used in protein extraction and purification protocols. It is designed to solubilize cellular proteins and maintain their native conformation. The buffer contains a combination of detergents, salts, and other components that facilitate the lysis of cells and the release of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Proteome profiler antibody arrays human xl cytokine array kit, by R&D Systems (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse or anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using radioimmunoprecipitation assay (ripa)
1
Cytokine Profiling of Extracellular Vesicles
2
Western Blotting of Cell Lysates
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Cells were dissociated with Accutase and rinsed with PBS twice before being lysed with RIPA (Rockland) in the presence of 1% of Halt Protease and Phosphatase Inhibitor Cocktail (Pierce). Protein concentration was determined by a BCA assay kit (Thermo Fisher) according to manufacturer’s instructions. Samples containing 30 µg of total protein were loaded onto pre-cast 10% Tris-Glycine SDS/PAGE gels (Invitrogen) under denaturing conditions and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk in TBST, the membrane was incubated with primary antibody (Table S1 ) overnight at 4 °C. The membrane was then washed, incubated with an anti-mouse/rabbit peroxidase-conjugated secondary antibody (Table S1 ) for 1 hr at room temperature or overnight at 4 °C, and developed by SuperSignal chemiluminescence (Pierce).
3
Radiation-Induced Protein Analysis in RAW264.7 Cells
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Primed RAW264.7 cells were harvested at an appropriate time point after IR exposure and lysed with radioimmunoprecipitation assay buffer (RIPA; Rockland, Limerick, PA, USA). The lysate was incubated for 20 min in ice and subsequently centrifuged at 13,000 rpm for 15 min at 4°C to remove cell debris, after which protein concentration was determined using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Pittsburgh, PA, USA) according to manufacturer instructions. We performed 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by probing of the membranes overnight at 4°C using primary antibodies against iNOS, COX-2, and glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling Technology, Danvers, MA, USA). The blots were then incubated with a secondary horseradish peroxidase (HRP)conjugated anti-mouse or anti-rabbit IgG antibody (Cell Signaling Technology) for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific).
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