Ab99702

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab99702 is a laboratory equipment product. It is a device used for a specific function in a laboratory setting. No further details about its intended use or performance can be provided in an unbiased and factual manner.

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Lab products found in correlation

8 protocols using ab99702

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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Western Blot Analysis of ECT2 and GAPDH

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Cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl, 100 mM 2-mercaptoethanol, 2% w/v SDS, 10% glycerol; pH 6.8). Proteins (100 µg) were separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, Chalfont, UK). Subsequently, the PVDF membrane was blocked with phosphate-buffered saline supplemented with 5% milk overnight at 4°C, and incubated with rabbit anti-ECT2 (1:100; ab123571) or rabbit anti-GAPDH (1:200; ab181602; both Abcam, Cambridge, UK) monoclonal antibodies at room temperature for 3 h, respectively. Following washing three times with phosphate-buffered saline and Tween 20 for 5 min, the PVDF membrane was incubated with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (1:10,000; ab99702; Abcam) at room temperature for 40 min. Super Signal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) was used to detect the signals according to the manufacturer's protocol. Relative protein expression was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA), and was represented as a density ratio compared with GAPDH.

Tan H., Wang X., Yang X., Li H., Liu B, & Pan P. (2016). Oncogenic role of epithelial cell transforming sequence 2 in lung adenocarcinoma cells. Experimental and Therapeutic Medicine, 12(4), 2088-2094.

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Western Blot Analysis of Wnt Pathway

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Cells were lysed on ice using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) and protein concentrations were determined using a bicinchoninic acid assay. Proteins (40 µg) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore) and incubated with 5% fat-free skim milk in TBS + 0.05% Tween 20, for 1 h at room temperature. Membranes were subsequently incubated overnight at 4°C with the following antibodies: Anti-CTNNB1 (rabbit monoclonal; 1:1,000; ab16051), anti-β-catenin (rabbit monoclonal; 1:1,000; ab32572), anti-TCF4 (rabbit polyclonal; 1:1,000; ab185736) or anti-β-actin (rabbit polyclonal; 1:1,000; ab8227) (all from Abcam, Cambridge, MA, USA). Secondary antibody incubations were performed with horseradish peroxidase-conjugated mouse anti-rabbit antibody (1:10,000; ab99702; Abcam) for 1 h at room temperature. Enhanced chemiluminescence substrate was used to visualize signals (EMD Millipore). β-actin was used as an endogenous protein for normalization. Relative band intensities were determined by densitometry using Quantity One 4.6.2 software (Bio-Rad Laboratories, Inc.).

Cao F., Zhan J., Chen X., Zhang K., Lai R, & Feng Z. (2017). miR-214 promotes periodontal ligament stem cell osteoblastic differentiation by modulating Wnt/β-catenin signaling. Molecular Medicine Reports, 16(6), 9301-9308.

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Western blot analysis of esophageal tissue

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Radio immunoprecipitation assay lysis buffer (C0481, Sigma-Aldrich) was used for total protein extraction.²³ (link) The protein sample was quantified using a bicinchoninic acid kit (Beyotime, Shanghai, P.R. China), separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Immunoblots were incubated with primary antibodies overnight at 4°C and then incubated with horseradish peroxidase-conjugated secondary antibodies (ab99702, Abcam, Cambridge, UK). Immunoreactive bands were visualized by enhanced chemiluminescence (Baomanbio, Shanghai, P.R. China), and protein expression was normalized against β-actin. Antibodies against the following proteins were used (all from Abcam): β-actin (ab115777), FOXO6 (ab48730), c-CASP3 (ab13847), USP (ab4080), JMJD3 (ab38113), HA (ab9110), FLAG (ab1162), and Myc (ab32072). The levels being reported were relative to the mean expression of three non-cancerous samples (three adjacent non-tumorous esophageal tissues or normal esophageal cells) for each experiment.

Li N., Zhao Z., Liu P., Zheng Y., Cai S., Sun Y, & Wang B. (2020). Upregulation of deubiquitinase USP7 by transcription factor FOXO6 promotes EC progression via targeting the JMJD3/CLU axis. Molecular Therapy Oncolytics, 20, 583-595.

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Western Blot Analysis of Lung Fibrosis

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Human lung tissues from controls, patients with IPF, and patients with RA-ILD or mouse lung fibroblasts were lysed by lysis buffer (C0481, Sigma-Aldrich Chemical Company, St. Louis, MO, United States) to extract total protein. Protein loading buffer was added to the supernatant. After boiling for 5 min, 20 μg of protein sample was electroporated onto a polyvinylidene fluoride membrane by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Millipore, Billerica, MA, United States). The membrane was added with Tris-buffered saline containing Tween 20 (TBST) diluted primary antibodies IL17RA (ab180904, 1:1,000, Abcam), HDAC3 (ab219376, 1:1,000, Abcam), and GAPDH rabbit anti (ab181602, 1:10,000, Abcam) for incubation overnight at 4°C. HRP-labeled secondary antibody (ab99702, 1:1,000, Abcam) was added and incubated for 1 h. Enhanced chemiluminescence (Shanghai Baoman Biotechnology Co., Ltd., Shanghai, China) was used for developing. GAPDH was used as an internal reference. The Image J was used to analyze the luminosity of each band, and the ratio of the value of the target protein to the internal reference was calculated.

Yuan H., Jiao L., Yu N., Duan H., Yu Y, & Bai Y. (2020). Histone Deacetylase 3-Mediated Inhibition of microRNA-19a-3p Facilitates the Development of Rheumatoid Arthritis-Associated Interstitial Lung Disease. Frontiers in Physiology, 11, 549656.

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Western Blot Analysis of EMT Markers

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Cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 6.8, 100 mM 2-ME, 2 %w/v SDS, 10 % glycerol). After centrifugation at 20,000×g for 10 min at 4 °C, proteins in the supernatants were quantified and separated with 10% SDS-PAGE. Then, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Bioscience, Buckinghamshire, USA), which was then incubated with PBS containing 5% milk overnight at 4 °C. The PVDF membrane was incubated with rabbit anti-human primary antibodies including SALL4 (polyclonal, 1:100, ab29112), E-cadherin (polyclonal, 1:50, ab15148), N-cadherin (polyclonal, 1:50, ab18203), Fibronectin (polyclonal, 1:200, ab2413), vimentin (monoclonal, 1:50, ab16700), MMP2 (polyclonal, 1:200, ab37150), MMP9 (polyclonal, 1:100, ab38898), and GAPDH (polyclonal, 1:100, ab181602) (all from Abcam, Cambridge, MA, USA) at room temperature for 3 h, respectively, and then with mouse anti-rabbit secondary antibody (monoclonal, 1:10000, ab99702, Abcam) at room temperature for 1 h. Super Signal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL, USA) was then used to detect signals, according to the manufacture's instruction. The relative protein expression was analyzed by Image-Pro plus software 6.0, represented as the density ratio versus GAPDH.

Zhou W., Zou B., Liu L., Cui K., Gao J., Yuan S, & Cong N. (2016). MicroRNA-98 acts as a tumor suppressor in hepatocellular carcinoma via targeting SALL4. Oncotarget, 7(45), 74059-74073.

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Western Blot Analysis of IGF-1R Expression

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Cells were solubilized in cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Inc., Shanghai, China). A bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Inc.) was used to determine the protein concentration according to the manufacturer's instructions. Protein (50 µg per lane) was separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.), which was incubated with PBS containing 5% milk (Mengniu, Beijing, China) overnight at 4°C. After washing with PBS (Thermo Fisher Scientific, Inc.) three times, the PVDF membrane was then incubated with rabbit anti-human IGF-1R monoclonal antibody (1:100 dilution; ab182408; Abcam, Cambridge, MA, USA) or rabbit anti-human GAPDH monoclonal antibody (1:200 dilution; ab9485; Abcam) at room temperature for 3 h. After washing with PBS for three times, the PVDF membrane was incubated with mouse anti-rabbit secondary antibody (1:5,000 dilution; ab99702; Abcam) at room temperature for 40 min. An enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) was then used to visualize the blots according to the manufacturer's instruction. Image-Pro plus software 6.0 (Media Cybernetics, USA) was used and the relative protein expression of IGF-1R was represented as the density ratio vs. GAPDH.

Wang Z., Zheng C., Jiang K., He J., Cao X, & Wu S. (2017). MicroRNA-503 suppresses cell proliferation and invasion in osteosarcoma via targeting insulin-like growth factor 1 receptor. Experimental and Therapeutic Medicine, 14(2), 1547-1553.

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Western Blot Analysis of Exosomal Proteins

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Isolated cells and exosomes were subjected to RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with the Protease Inhibitor Cocktail (Roche). Purified proteins were separated in 6%, 10%, or 12% SDS-PAGE (120 V for stacking gel and 160 V for separation gel) and then transferred to a nitrocellulose membrane in an ice bath. The nitrocellulose membrane was blocked with 5% bovine serum albumin for 1 h and then incubated overnight with primary antibodies at 4 °C. Antibodies used were mouse anti-CD63 (Abcam, ab59479), rabbit anti-CD9 (Abcam, ab92726), mouse anti-TSG101 (Santa, sc-7964), rabbit anti-GM130 (Abcam, ab30637), rabbit anti-Cltc (Cell Signaling Technology, #4796), rabbit anti-GAPDH (Abcam, ab181602). The membrane was then incubated with secondary antibodies (rat anti-mouse (Abcam, ab99632), mouse anti-rabbit (Abcam, ab99702)) for 1 h at room temperature and visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire UK).

Wan Z., Zhao L., Lu F., Gao X., Dong Y., Zhao Y., Wei M., Yang G., Xing C, & Liu L. (2020). Mononuclear phagocyte system blockade improves therapeutic exosome delivery to the myocardium. Theranostics, 10(1), 218-230.

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