For Western blot analysis, the cells were treated with ZnO NPs (30 μg/mL) for 12 h. The harvested cell pellets were incubated in RIPA lysis buffer in the presence of a protease inhibitor. Protein concentrations were measured using BCA Protein Assay Reagent (Pierce, Rockford, IL, US). The cell lysates were then analyzed for protein content using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was probed with antibodies to determine the level of protein expression. The following primary antibodies were used: anti-γ-H2AX (ab26350; Abcam), anti-Rad51 (ab88572; Abcam), anti-Caspase-9 (ab202068; Abcam), anti-p53 (ab1431; Abcam), anti-LC3 (ab51520; Abcam), anti-BAX (#2772; Cell Signaling Technology, Boston, MA, USA), anti-Bcl2 (YT0470; ImmunoWay, SuZhou, China), and anti-β-action (ab8227; Abcam).
Ab1431
Manufactured by Abcam
Sourced in United States
Ab1431 is a laboratory instrument used for the detection and quantification of proteins. It employs an electrochemical detection method to provide accurate and sensitive measurements. The core function of Ab1431 is to analyze protein samples and generate reliable data for scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab8226, by Abcam (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ab88572, by Abcam (2 mentions)
Ab26350, by Abcam (2 mentions)
Ab51520, by Abcam (2 mentions)
Ab202068, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab32148, by Abcam (2 mentions)
Bca protein assay, by Beyotime (2 mentions)
Ab2302, by Abcam (2 mentions)
Ab128908, by Abcam (2 mentions)
Gtx56293, by GeneTex (2 mentions)
Ab9485, by Abcam (2 mentions)
Ab469, by Abcam (2 mentions)
Ab179800, by Abcam (2 mentions)
Ab75972, by Abcam (2 mentions)
Ab37185, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
29 protocols using ab1431
1
Western Blot Analysis of ZnO NP-Treated Cells
2
Esophageal Squamous Cancer Cell Line Cultivation
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Esophageal squamous cancer cell line KYSE450 was obtained from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were cultured in RPMI 1640 (21875-034, Gibco, UK) supplemented with 10% FBS (16000-044, Gibco, USA), 100 U/mL of penicillin and 100 µg/mL streptomycin (15140-122, Gibco, USA) at 37°C and 5% CO2.
The primary antibodies and drugs used in this study were as follows: PDHA1 (ab 177461, Abcam, Shanghai, People’s Republic of China); ANG2 (bs-0677R, Bioss, Beijing, People’s Republic of China); VEGFRA (GB11034B; Servicebio, Wuhan, People’s Republic of China); P53 (af0879, Affinity Biosciences, USA); Phospho p53 (S15) (ab1431, Abcam, Shanghai, People’s Republic of China); ABCG2 (12-888-42, Invitrogen, USA); CD31 (GB13063; Servicebio, Wuhan, People’s Republic of China); GAPDH (AF5718, R&D Systems, USA); ACTIN (GB12001; Servicebio, Wuhan, People’s Republic of China); Docetaxel (S1148, Selleckchem, USA); Paclitaxel (S1150, Selleckchem, USA).
The primary antibodies and drugs used in this study were as follows: PDHA1 (ab 177461, Abcam, Shanghai, People’s Republic of China); ANG2 (bs-0677R, Bioss, Beijing, People’s Republic of China); VEGFRA (GB11034B; Servicebio, Wuhan, People’s Republic of China); P53 (af0879, Affinity Biosciences, USA); Phospho p53 (S15) (ab1431, Abcam, Shanghai, People’s Republic of China); ABCG2 (12-888-42, Invitrogen, USA); CD31 (GB13063; Servicebio, Wuhan, People’s Republic of China); GAPDH (AF5718, R&D Systems, USA); ACTIN (GB12001; Servicebio, Wuhan, People’s Republic of China); Docetaxel (S1148, Selleckchem, USA); Paclitaxel (S1150, Selleckchem, USA).
3
Immunoblot Analysis of Cell Signaling
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Cells were harvested and dissolved in RIPA lysis buffer, and the protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Beyotime Biotechnology, Jiangsu, China). Whole cell lysates were fractionated and transferred to PVDF membranes by an electroblot apparatus. Membranes were incubated at 4 °C overnight with specific primary antibodies. Bands were visualized with an ECL detection reagent (Beyotime). The densitometric quantification was analysed with a β-actin control using Image Lab software (Bio-Rad, Hercules, CA, USA). All antibodies used in this study were obtained from Santa Cruz (CA, USA) and Abcam (Cambridge, UK). The primary antibodies were rabbit monoclonal anti-ALKBH5 (ab195377, Abcam), mouse monoclonal anti-PER1 (sc-398,890, Santa Cruz), mouse monoclonal anti-p-ATM (Ser-1981; sc-47,739, Santa Cruz), rabbit monoclonal anti-p-CHK2 (Thr-68; ab32148, Abcam), rabbit polyclonal anti-p-CDC25C (Ser216; ab47322, Abcam), rabbit monoclonal anti-p-P53 (Ser-15; ab1431, Abcam), mouse monoclonal anti-P21 (sc-71,811, Santa Cruz), mouse monoclonal anti-CYCLIN B1 (ab72, Abcam), rabbit polyclonal anti-p-CDK1 (Tyr15; ab47594), mouse monoclonal anti-CDK1 (A17, Abcam) and rabbit polyclonal anti-β-ACTIN (ab8227, Abcam).
4
Renal Protein Extraction and Western Blot
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Proteins were extracted from renal tissue using an ice-cold lysis buffer, and the protein concentrations were quantified using a BCA assay kit (PC0020; Solarbio). SDS-PAGE was used to separate equal amounts of protein. The blocked membrane was incubated using anti-Bax (1 : 500; WL01637, WanleiBio), anti-P-p53 (1 : 2,000; ab1431, Abcam), anti-PUMA (1 : 3,000; ab9643, Abcam), anti-caspase-3 (1 : 2,000; 9661, CST), anti-Nrf2 (1 : 500; ab89443, Abcam), anti-HO-1 (1 : 500; WL01637, WanleiBio), anti-OCT2 (1 : 500; ab243153, Abcam), anti-MRP2 (1 : 500; ab203397, Abcam), and anti-β-actin (1 : 1,000; WL01372, WanleiBio) antibodies overnight at 4°C. Then, the membranes were incubated with a suitable horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. Finally, an ECL western blotting substrate was used to visualize the immunoblots.
5
Protein Extraction and Analysis of UBE2S Knockdown in A549 Cells
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A549 cells expressing shCtrl or shUBE2S lentivirus were cultured for 48 hours and used for protein isolation. In brief, culture medium was removed and cells were washed by PBS. Lysis buffer (100 mM Tris-HCl, pH = 7.4 l 0.15 M NaCl; 5 mM EDTA, pH = 8.0; 1% Triton X100; 5 mM DTT; 0.1 mM PMSF) was added into 6 cm plates to extract total protein. BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used for protein quantification. A total of 30 μg protein lysis were loaded into SDS-PAGE electrophoresis, and subsequently transferred onto PVDF transmembrane. Membranes were blocked with 5% skim milk for 1 hour at room temperature, and then incubated with primary antibodies overnight at 4°C. After washing membranes with PBST, HRP conjugated secondary antibodies were added. ECL-Plus kit (Amersham Biosciences, Pollards Wood, UK) was used to detect immunoactivity. Primary antibody against UBE2S (ab197945), p53 (ab1431) and NUPR1 (ab6028) were purchased from Abcam. Primary antibody against ILK (#3856) and Cleaved PARP1 (#5625) were purchased from Cell Signaling Technology. GAPDH primary antibody (SC-32233) and secondary antibodies were obtain from Santa Cruz.
6
Immunohistochemical Analysis of Tumor Markers
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Mice tumour tissues were collected and made into 5‐μm paraffin‐embedded sections. Sections were dewaxed at 67°C, and then endogenous peroxidase was inactivated with 3% hydrogen peroxide methanol solution. Non‐specific antigens were blocked with 10% goat serum at 37°C for 1 hour. After washing thrice with PBS, sections were incubated at 37°C for 1.5 hours with primary antibodies, and then incubated with secondary antibody for 1 hour at room temperature. Then sections were washed with PBS and incubated with 3, 3′‐Diaminobenzidine (DAB; Sigma‐Aldrich) for 5 minutes in a dark room. After being washed with PBS, sections were then counterstained for 1 minute with hematoxylin (Sigma‐Aldrich). Permount™ Mounting Medium (Abcam) was utilized to mount the sections. Primary antibodies used were as listed below: rabbit anti‐SERPINE1 (5 μg/mL, ab66705; Abcam), rabbit anti‐p53 (20 μg/mL, ab1431; Abcam), rabbit anti‐MDM2 (1:100, ab131355; Abcam), rabbit anti‐cleaved Caspase3 (10 μg/mL, ab2302; Abcam) and rabbit anti‐ki67 (1:50, ab8191; Abcam). Secondary antibody was HRP‐labelled goat anti‐rabbit IgG (1:2000, ab205718; Abcam).
7
Immunofluorescent Staining of Testis Tissues
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Testis tissues or TM3 cells were fixed and processed for immunofluorescent or immunohistochemical staining as described previously [35 (link)]. The primary antibodies included anti-FOXO4 (ab128908, 1:100; Abcam, USA), anti-FOXO4 (sc-373877, 1:100; Santa Cruz, USA), anti-StAR (#8449, 1:100; Cell Signaling Technology, USA), anti-CYP11A1 (GTX56293, 1:100; Gene Tex, USA), anti-3β-HSD (sc-100466, 1:100; Santa Cruz, USA), and anti-Ser15-phospho-p53 (ab1431, 1:100; Abcam, USA) antibodies. Images were captured using a Leica confocal microscope.
8
Immunohistochemical Staining of MMP-9 and p53
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Routine immunohistochemical staining (SP method) was used in the current study. The primary antibody for MMP-9 was rabbit polyclonal to MMP-9 antibody (ab38898, Abcam plc, Cambridge, MA, USA); and rabbit polyclonal to p53 antibody for p53 primary antibody (ab1431, Abcam plc); the negative control antibody was PBS (ab154878, Abcam plc); the remaining steps were the same as described before. The cells having brown yellow grain coarse particle distribution or brown yellow fine particle distribution were defined as positive. The optical density of the positive reaction was determined by the TONGJI HPIAS-1000 color pathological image analysis system (Qingping image technology Ltd., Wuhan, CHN).
9
Western Blot Analysis of Protein Levels
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Equal amounts of protein were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5%), blotted to a nitrocellulose membrane and then blocked in phosphate-buffered saline (PBS) with 0.05% (v/v) Tween-20 (T-PBS) containing 1% (w/v) bovine serum albumin. Each blot was incubated overnight at 4 °C with primary antibodies. Primary antibodies for hnRNP H1 (ab10374) and p53 (phospho S15) (ab1431) were purchased from Abcam, primary antibody for SGPL1 (AF5535) was purchased from R&D Systems (Minneapolis, MN, USA) and primary antibody for p53 (506135) was purchased from Calbiochem. The blots were then washed in T-PBS, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (R&D Systems), washed in T-PBS and then developed using a Super-Signal West Pico enhanced chemiluminescence system (Thermo Fisher Scientific). The averaged protein expression was normalized to actin expression (BD Transduction Laboratories, Lexington, KY, USA).
10
Western Blot Analysis of Apoptotic Markers
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SW480 cells were collected at the treatment time-points with reagents as suggested in each experiment. Cells were lysed with RIPA buffer (Thermo Fisher Scientific) and 15% electrophoresis (Thermo Fisher Scientific) was conducted under 90 V for 2 h. After undergoing electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. Primary antibodies, rabbit polyclonal anti-fibronectin antibody (1:500), rabbit polyclonal caspase-3 antibody (1:500, ab2302), rabbit polyclonal NF-κB antibody (1:500, ab7971), rabbit polyclonal p53 antibody (1:500, ab1431), rabbit polyclonal PARP (1:500, ab6079), rabbit polyclonal Bax antibody (1:500, ab53154), rabbit polyclonal cytochrome c antibody (1:500, ab90529) and rabbit polyclonal GAPDH antibody (1:500, ab9485) (all from Abcam) were incubated with the membranes for 24 h at 4°C. After washing with TBST buffer three times, the secondary antibody goat anti-rabbit IgG H&L (HRP) was incubated at room temperature for 1 h. Immunostaining was carried out using DAB Plus substrate and chemiluminescence system (Amersham Biosciences, Freiburg, Germany). The results were analyzed using chemiluminescence Molecular Imager® ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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