A25012

Manufactured by Abbkine

Sourced in United States

A25012 is a laboratory equipment product. It is a device designed for use in scientific research and analysis. The core function of the product is to perform a specific task or operation within a laboratory setting. No further details or interpretation about its intended use are provided.

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Lab products found in correlation

3 protocols using a25012

Osteosarcoma Cell Lines and Antibody Specifications

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The human osteosarcoma cell lines MG63, MNNG-HOS and Saos-2 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human osteosarcoma cell line U-2OS was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were cultured following the ATCC protocols. Standardized culture conditions had been described previously [18 (link)].
The antibodies used were S1PR3 (ab126622; Abcam, Cambridge, UK), YAP (ab52771; Abcam, Cambridge, UK), p-YAP (ab76252; Abcam, Cambridge, UK), c-Myc (ab32072; Abcam), Ki67 (GB13030; Servicebio, Wuhan, China), β-actin (M1210-2; Hua'an Biology, Chuzhou, China), GAPDH (bsm-33033M; Bioss, Beijing, China), anti-rabbit IgG light chain (ab99697, Abcam), anti-rabbit IgG heavy chain (ab99702, Abcam), and anti-mouse IgG light chain (A25012, Abbkine, CA, USA).

Shen Y., Zhao S., Wang S., Pan X., Zhang Y., Xu J., Jiang Y., Li H., Zhang Q., Gao J., Yang Q., Zhou Y., Jiang S., Yang H., Zhang Z., Zhang R., Li J, & Zhou D. (2018). S1P/S1PR3 axis promotes aerobic glycolysis by YAP/c-MYC/PGAM1 axis in osteosarcoma. EBioMedicine, 40, 210-223.

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Protein Interaction Verification Techniques

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Both Western blot and co-immunoprecipitation were performed as described previously [25 (link)]. For whole-cell extracts, cells were lysed with 20 mM Tris·HCl, pH 8.0, 100 mM NaCl, 0.5% NP-40, and 1 mM EDTA (NETN buffer) supplemented with Micrococcal Nuclease (1:5000, M0247S, NEB) on ice for 15 min. Cell lysates were boiled with 5× SDS loading buffer, separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and immunoblotted with indicated antibodies. For co-IP experiments, cells were lysed with NETN buffer for 15 min on ice, followed by centrifugation at 12,000 rpm for 10 min at 4 °C. Supernatants were then transferred into new Eppendorf tubes and incubated with either Flag beads (L00425, GenScript) for 4 h or Protein A/G plus agarose beads (sc-2003, Santa Cruz) conjugated with indicated antibodies overnight at 4 °C with rotation. Beads were subsequently washed three times with NETN buffer and boiled with 1× SDS loading buffer. To avoid the noise of light/heavy chains, the secondary antibodies used for the immunoprecipitation assays including mouse anti-rabbit IgG LCS (A25022, 1:5000) and goat anti-mouse IgG LCS (A25012, 1:5000) were obtained from Abbkine.

Zhang L., Lu X., Xu Y., La X., Tian J., Li A., Li H., Wu C., Xi Y., Song G., Zhou Z., Bai W., An L, & Li Z. (2023). Tumor-associated macrophages confer colorectal cancer 5-fluorouracil resistance by promoting MRP1 membrane translocation via an intercellular CXCL17/CXCL22–CCR4–ATF6–GRP78 axis. Cell Death & Disease, 14(9), 582.

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Western Blot Analysis of FTO and NOD1

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Cellular lysates were generated by using 1×SDS-PAGE loading buffer. Proteins were extracted from cells and measured with the BCA Protein Assay kit (Beyotime; P0012S), then subjected to SDS-PAGE (8%) gel and transferred to PVDF (Millipore; ISEQ00010) membranes by semidry blotting (Bio-Rad Trans Blot Turbo System). The membranes were blocked with 5% BSA. Protein was blotted with different antibodies. The antibody against FTO was diluted at 1: 500 (Beyotime; AF6936); The antibody against NOD1 was diluted at 1: 1000 (Customized by Genscript); The antibody against NOD1 of human was diluted at 1: 1000 (Absin; abs115515); Anti-Flag (Beyotime; AF519) and anti-Tubulin (Beyotime; AT819) monoclonal antibody were diluted at 1: 2,000; and HRP-conjugated anti-rabbit IgG (Abbkine; A25022) or anti-mouse IgG (Abbkine; A25012) at 1: 5,000. The results were the representative of three independent experiments. The immunoreactive proteins were detected by using WesternBright^TM ECL (Advansta). The digital imaging was performed with a cold CCD camera.

Geng S., Zheng W., Zhao Y, & Xu T. (2022). FTO promotes innate immunity by controlling NOD1 expression via m6A-YTHDF2 manner in teleost. iScience, 25(12), 105646.

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