Ovarian tissues were homogenized in RIPA buffer (Thermo Scientific, Rockford, IL, USA) and centrifuged at 12,000 × g for 20 min. The protein content of samples was determined using a BCA protein assay kit (Bio-Rad Laboratories, Carlsbad, CA). Aliquots of each sample were separated via 12% SDS-PAGE and then transferred onto a nitrocellulose membrane, which was blocked with non-fat milk for 1 h. The membranes were probed overnight at 4°C with antibodies against the following proteins: HO-1 (ab68477; 1:2000; Abcam); NRF2 (ab92946; 1:3000; Abcam), TNF-α (SAB5700627; 1:1000; Sigma), IL-1β (AB1413-I; 1:4000; Sigma), IL-6 (SAB5700632; 1:2000; Sigma); MVH (ab13840; 1:3000; Abcam); OCT4 (ab184665; 1:2000; Abcam); Ki67 (ab16667; 1:4000; Abcam), PCNA (mAb2586; 1:3000; Cell Signaling Technology), ATM (ab199726; 1:2000; Abcam), RAD51 (ab176458; 1:3000; Abcam), and GAPDH (ab181602; 1:3000; Abcam). After washing with PBST buffer solution three times, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Beyotime Institute of Biotechnology) for 1 h at 25 °C, and the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific Inc.). The blots were scanned and normalized to GAPDH for quantification.
Ab199726
Manufactured by Abcam
Sourced in United States
Ab199726 is a laboratory equipment product from Abcam. It serves as a core laboratory tool for researchers.
Automatically generated - may contain errors
Lab products found in correlation
Ab26350, by Abcam (2 mentions)
Ab81292, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab184665, by Abcam (1 mentions)
Ab13840, by Abcam (1 mentions)
Ab68477, by Abcam (1 mentions)
Ab92946, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Sab5700632, by Merck Group (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
Ab124762, by Abcam (1 mentions)
Mir 1205 mimic, by RiboBio (1 mentions)
Ab32389, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Gapdh sc 365062, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Ab7960, by Abcam (1 mentions)
5 protocols using ab199726
1
Proteomic Analysis of Ovarian Tissues
2
Docetaxel and miR-1205 Mechanistic Study
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Docetaxel (DOC) was purchased from MCE (Beijing, China). miR-1205 mimic was purchased from Ribobio (Guangzhou, China). Chemicals were purchased from Sigma-Aldrich (St Louis, USA). Antibodies were purchased from Santa Cruz Biotechnology (Dallas, USA) and Abcam (Cambridge, USA). The antibodies used in this study were as follows: DNAJB1 (sc-398766, 1:1000 for western blotting, 1:100 for Co-IP; Santa Cruz), p53 (ab32389, 1:1000 for western blotting, 1:200 for Co-IP; Abcam), p63 (1:1000 for western blotting, 1:200 for Co-IP) (ab124762; Abcam), ATM (ab199726, 1:1000; Abcam), Bax (ab32503, 1:1000; Abcam), GAPDH (sc-365062, 1:500; Santa Cruz). Other chemicals and reagents were purchased from Beyotime (Nantong, China) and Sangon (Shanghai, China).
3
Western Blot and IHC Protocol
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Western blotting and immunohistochemistry were performed according to standard procedures50 (link). The following antibodies were used: anti-HBX (ab39716, Abcam, Cambridge, MA, USA), anti-P-CHK2 (2197S, Cell Signaling Technology, Danvers, MA, USA), anti-CHK2 (ab47433, Abcam), anti-ATM (ab199726, Abcam), anti-P21 (ab7960, Abcam), anti-P53 (BS1913, Bioworld, Minnesota, USA), anti-γH2AX (ab26350, Abcam), anti-P-CHK1 (2348S, Cell Signaling Technology), anti-CHK1 (10362-1-AP, Proteintech), anti-CDK2 (SC-6248, Santa Cruz Biotechnology, CA, USA), anti-Cyclin D1 (SC-718, Santa Cruz Biotechnology), anti-GAPDH (SC-47724, Santa Cruz Biotechnology), HRP-Ms (#7074, Cell Signaling Technology), and HRP-Rb (#7076, Cell Signaling Technology).
4
Protein Expression Analysis in OS Cells
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Protein samples were collected from OS cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) according to the manufacturer’s instructions. A total of 30 μg protein was loaded in each well, and 10% SDS-PAGE was performed. Then, proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen), which were subsequently blocked with 5% skim milk for 1 h, followed by incubation with primary antibodies including EXO1 (ab155553; Abcam, Cambridge, USA), ATM (ab199726; Abcam), p-ATM (ab81292; Abcam), ATR (#13934S; CST, Danvers, USA), p-ATR (#30632S; CST), p-CHK1 (#12302; CST), FBXO32 (ab168372; Abcam), γ-H2A.X (ab26350; Abcam), HA (AF5057; Beyotime), Flag (AF5051; Beyotime), β-Actin (#2D4H5; Proteintech, Chicago, USA), P53 (#60283-2-Ig; Proteintech), p-P53 (#67826-1-Ig; Proteintech), and CHK1 (#60277-1-Ig; Proteintech) overnight at 4°C. After being washed with Tris-buffered saline with Tween-20 (TBS-T) three times, PVDF membranes were incubated at 37°C for 1 h with the goat anti-mouse IgG(H+L) HRP (70-GAM0072; MULTISCIENCES, Hangzhou, China) or goat anti-rabbit IgG(H+L) HRP (70-GAR0072; MULTISCIENCES) as corresponding secondary antibody. Finally, chemiluminescence (ECL) luminous fluid (Beyotime) was added onto the PVDF membrane, and protein bands were detected using a chemiluminescence imaging instrument (Bio-Rad, Hercules, USA).
5
Western Blot Analysis of DNA Damage Response
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Mouse kidneys and HK-2 cells were lysed with cold lysis buffer (0.25 M NaCl, 50 mM Tris-HCl pH 7.4, 0.5% NP 40, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1% cocktail, and 1 mM PMSF). BCA reagent was used to determine the total protein concentration. The separated proteins were then electrophoretically transferred to a polyvinylidene fluoride membrane. After blocking with 5% fat-free milk in PBST for 2 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C. After being washed three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 60 min. Finally, an ECL substrate (sc2048, Santa Cruz) was used to visualize the immunoreactive bands. The main antibodies used for Western blot analysis included PLK3 (4896S, 1 : 1000, Cell Signaling), PLK3 (DF4471, 1 : 1000, Afftiny), ATM (ab199726, 1 : 1000, Abcam), p-ATM (ab81292, 1 : 1000, Abcam), P53 (ab179477, 1 : 1000, Abcam), P53 (YT3528, 1 : 1000, Immunoway), p-P53 (9287S, 1 : 1000, Cell Signaling), rH2AX (YP0218,1 : 1000, Immunoway), Bcl-2 (BF9103, 1 : 1000, Affinity), and Bax (GB12-690, 1 : 1000, Servicebio); the relative expression of the targeted protein was normalized to that of β-actin (AC026, 1 : 5000, ABclonal).
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