ChIP assays were performed as previously described 15 (link). Briefly, cells were crosslinked with 1% formaldehyde, then glycine was used to quench the cross-linking reaction. The cells were lysed in SDS buffer supplemented with protease inhibitor cocktail. Chromatin DNA was splintered into approximately 300 base-pair fragments by ultrasonication, which were then subjected to IP with 10 μg IgG (ab172730, Abcam), or 10 μg RORα (ab256799, Abcam). Washing with low and high-salt-concentration wash buffers, the DNA fragments were de-crosslinked under high-salt conditions. The QIAquick PCR purification kit (Vazyme) was used to purify DNA, followed by qRT-PCR.
Ab256799
Manufactured by Abcam
Sourced in United Kingdom
Ab256799 is a laboratory product designed for research purposes. It serves as a tool for conducting scientific experiments. The core function of this product is to facilitate data collection and analysis in a research setting. No further details or interpretation about its intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab92494, by Abcam (2 mentions)
Ab109250, by Abcam (2 mentions)
Ab181557, by Abcam (2 mentions)
Ab172730, by Abcam (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab181603, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Sangon (1 mentions)
Ecl luminescence reagent, by Sangon (1 mentions)
Ab32124, by Abcam (1 mentions)
Sc 7273, by Santa Cruz Biotechnology (1 mentions)
Ab32369, by Abcam (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Ab236630, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Ab275377, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab92742, by Abcam (1 mentions)
Ab126629, by Abcam (1 mentions)
Ab39638, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
7 protocols using ab256799
1
Chromatin Immunoprecipitation (ChIP) Protocol
2
Western Blot Analysis of Apoptosis Regulators
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Radioimmunoprecipitation assay lysis buffer (Sangon) was applied for total protein extraction, followed by Western blot detection referring to the issued description.16 (link)
The primary Abs targeting B-cell lymphoma-2 (Bcl-2; ab32124, 1:1000), Bcl-2-associated X (Bax; ab182733, 1:1000), RORA (ab256799, 1:1000), GAPDH (ab181603, 1:1000) were provided by Abcam (Cambridge, UK). Goat Anti-rabbit IgG H&L (HRP) secondary Ab (Abcam, ab205718, 1:5000) was incubated at 25°C for 1 h, followed by analysis of protein bands through ECL luminescence reagent (Sangon) and Image J software (NIH, Bethesda, MD, USA).
The primary Abs targeting B-cell lymphoma-2 (Bcl-2; ab32124, 1:1000), Bcl-2-associated X (Bax; ab182733, 1:1000), RORA (ab256799, 1:1000), GAPDH (ab181603, 1:1000) were provided by Abcam (Cambridge, UK). Goat Anti-rabbit IgG H&L (HRP) secondary Ab (Abcam, ab205718, 1:5000) was incubated at 25°C for 1 h, followed by analysis of protein bands through ECL luminescence reagent (Sangon) and Image J software (NIH, Bethesda, MD, USA).
3
Immunoprecipitation of PPAR-γ, HDAC3, and RORα
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Phosphate-buffered saline was used to wash the cells before lysing them in lysis buffer (200 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 0.5% NP40). After that, the lysates were mixed with protein A beads (CST, Waltham, MA; 70024) and incubated overnight at 4°C with primary antibodies against anti-PPAR-γ (sc-7273; Santa Cruz Biotechnology, Dallas, TX), anti-HDAC3 (ab32369; Abcam, Boston, MA), anti-RORα (ab256799; Abcam), or the corresponding normal IgG (acts as negative control). To remove unbound proteins, the beads were washed twice with lysis buffer. To elute the bound proteins, the beads were resuspended in sample buffer, boiled for 3 minutes at 95°C, and immunoblotted.
4
Exosome Protein Profiling by Western Blot
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Cell lysates or exosomes were extracted. 12% SDS-PAGE was implemented for protein separation. Then proteins were shifted to PVDF membranes. Followed by sealing with 5% defatted milk, the primary antibody TSG101 (Abcam, ab125011, 1:2000), CD9 (Abcam, ab236630; 1:1000), ALIX (Abcam, ab275377, 1:1000), GAPDH (Abcam, ab9485, 1:2500), NANOG (Abcam, ab109250, 1:2000), SOX2 (Abcam, ab92494, 1:2000), OCT4 (Abcam, ab181557, 1:1000) and RORA (Abcam, ab256799, 1:1000) were cultivated with the membranes overnight at 4 °C. Subsequently, a secondary antibody was cultivated with the membranes and the bands were visualized with enhanced chemiluminescence using an ECL kit (Beyotime, Shanghai, China). The relative densities of protein bands were analyzed by ImageJ (v1.8.0; National Institutes of Health).
5
Immunohistochemical Evaluation of Stem Cell Markers
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Sections (4-µm thickness) cut from paraffin tissue were dewaxed with xylene, soaked with 0.01 mol/L sodium citrate and heated in the microwave. Then the sections were hatched with primary antibodies against Ki-67 (Abcam, ab92742, 1:500), NANOG (Abcam, ab109250, 1:200), OCT4 (Abcam, ab181557, 1:1000), SOX2 (Abcam, ab92494, 1:100) and RORA (Abcam, ab256799, 1:800). Next, the sections were hatched with HRP conjugated secondary antibody. The reaction was developed by diaminobenzidine and redyed with hematoxylin and observed via a microscope (Olympus, Tokyo, Japan).
6
IHC Analysis of Tumor Biomarkers
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Tumor biopsies were fixed with formalin, embedded in paraffin and cut into 5-μm sections. Samples were then deparaffinized and dehydrated, and subsequently rehydrated with demineralized water. The antigen was extracted by an immunohistochemical method utilizing microwave pretreatment. IHC staining was performed as previously described using following antibodies from Abcam: anti-RORα (ab256799), anti-ECM1 (ab126629) and anti-VEGFR2 (ab39638). Goat anti-rabbit horseradish peroxidase-conjugated antibody was used as secondary antibody (ab205718, Abcam). Proteins were visualized in situ with a 3, 3ʹ-diaminobenzidine reaction solution.
7
Protein Expression Analysis of Chondrocytes
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Cartilage tissues were weighed and then lysed by RIPA lysate on the ice for 15 min. Then, samples were centrifuged with 12,000 g/min for 15 min at 4°C. The supernatant was removed. Cultured chondrocytes were washed and lysed by RIPA. BCA protein assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to quantify the protein levels. The protein samples were electrophoresed in SDS-PAGE to separate protein bands. Proteins were transferred from gel onto PVDF membrane, blocked with 5% non-fat dry milk for 2 h. The PVDF membrane was incubated with monoclonal rabbit antibodies specific for MMP-3 (ab52915, Abcam), MMP-13 (MAB13426, Sigma-Aldrich), SOX9 (ab185966, Abcam), Collagen II (ab188570, Abcam), CH25H (sc-293256, SantaCruz), CYP7B1 (ABN182, Sigma-Aldrich) and RORα (ab256799, Abcam) overnight at 4°C. On the next day, membranes were washed and then incubated with second antibody for 2 h. The bands were visualized by ECL method (Beyotime Biotechnology Co., Ltd., Shanghai, China) and the overall gray value of protein bands (average gray value x gray value area) was quantified with Photoshop CS5 software to calculate the target protein relative value (target protein gray value/internal reference overall gray value).
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