To extract whole cell lysate, cells were harvested using RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). Cell lysates were subjected to SDS-PAGE and proteins transferred onto a nitrocellulose membrane (Osmonics Inc.). The membrane was probed with primary antibodies (described above) followed by a secondary antibody conjugated with fluorescent dye and detected using the Odyssey detecting system (LI-COR Bioscience).
Odyssey detecting system
Manufactured by LI COR
Sourced in Germany
The Odyssey Detecting System is a versatile imaging platform designed for a range of life science applications. It offers high-sensitivity detection and quantification of fluorescent and chemiluminescent signals on gels, blots, and microplates. The system combines state-of-the-art optics, illumination, and detection technologies to provide accurate and reliable data.
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Lab products found in correlation
4 protocols using odyssey detecting system
1
Whole Cell Lysate Extraction and Western Blot
2
Chromatin Immunoprecipitation Protein Analysis
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Whole cell lysates were harvested using radio-immunoprecipitation assay (RIPA) buffer composed of 50 mM trisaminomethane hydrochloride (Tris-HCl) pH7.5, 150 mM sodium chloride (NaCl), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1.0 mM EDTA, 1.0 mM sodium orthovanadate, and 1x protease inhibitor cocktail. Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA). The membrane was probed with ChIP-grade H3K9me1 (ab9045, Abcam, Cambridge, MA), H3K9me2 (07-441, Sigma EMD Millipore, Darmstadt, Germany), KDM3A (A301-539A, Bethyl Laboratories, Montgomery, TX), or ACTB (A5441, Sigma EMD Millipore, Darmstadt, Germany) antibodies followed by a secondary antibody conjugated to fluorescent dye, and blots were imaged using the odyssey detecting system (LI-COR Biotechnology, Bad Homburg, Germany).
3
Immunoblotting for Androgen Receptor Quantification
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Whole cell lysates were harvested using radio-immunoprecipitation assay (RIPA) buffer composed of 50 mM trisaminomethane hydrochloride (Tris-HCl) pH 7.5, 150 mM sodium chloride (NaCl), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1.0 mM EDTA, 1.0 mM sodium orthovanadate, and 1x protease inhibitor cocktail. Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins transferred to a nitrocellulose membrane (GE Healthcare Life Sciences). The membrane was probed with an AR antibody (Sigma EMD Millipore, PG21, 06-680, Darmstadt, Germany) or actin antibody (Sigma EMD Millipore, A2066, Darmstadt, Germany) followed by a secondary antibody conjugated to fluorescent dye, and blots were imaged using the odyssey detecting system (LI-COR Biotechnology).
4
Western Blot Analysis of Inflammasome Proteins
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Cells (1 × 106) were preincubated at 37°C under 5% CO2 for 12 h and then treated by appropriate experimental reagents. Cells were then harvested on ice, washed three times with cold PBS, and suspended in 500 μL lysis buffer supplemented with protease inhibitors. After incubation on ice for 30 min, cell extracts were centrifuged at 14,000 rpm for 15 min at 4°C to isolate total cell proteins, which were quantified using a BCA protein assay kit. Proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes (Pierce, IL, USA) before hybridization with the appropriate detection antibodies (XO, pro-IL-1β, IL-1β, NLRP3, ASC, procaspase-1, and caspase-1). GAPDH was used to correct for differences in loading of the proteins and for normalization of the densitometric values of immunoblot signals obtained from three separate experiments using LI-COR Odyssey detecting system.
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