Protein samples were extracted from myocardial tissue and H9C2 cells. Protein concentrations were determined using the BCA method to ensure consistent loading of each sample. Proteins were separated using SDS-PAGE, and then transferred to 0.45-μm PVDF membranes. After blocking with skim milk for 1 hour, membranes were incubated with antibodies against Bcl-2 (ab32124), Bax (ab53154), p53 (ab32389), LC3B(ab63817), SQSTM1/62 (ab91526), ATG5 (ab53154), Beclin1 (ab62557), or β-actin (ab8226) (antibodies purchased from Abcam Biotechnology, USA) at 4 °C overnight. After labeling with secondary antibodies, the target protein was observed using enhanced chemical luminescence.
Ab53154
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab53154 is a laboratory equipment product manufactured by Abcam. It serves as a core functional component for research applications. The detailed specifications and intended use of this product are not available in this response.
Automatically generated - may contain errors
Lab products found in correlation
Ab196495, by Abcam (26 mentions)
Pvdf membrane, by Merck Group (16 mentions)
Ab205718, by Abcam (13 mentions)
Ab59348, by Abcam (12 mentions)
Ab9485, by Abcam (11 mentions)
Ab2302, by Abcam (9 mentions)
Ripa lysis buffer, by Beyotime (9 mentions)
Ab13847, by Abcam (9 mentions)
Ab8227, by Abcam (8 mentions)
Ab181602, by Abcam (7 mentions)
Ab8805, by Abcam (6 mentions)
Ab8226, by Abcam (5 mentions)
Ab182858, by Abcam (5 mentions)
Ripa buffer, by Beyotime (5 mentions)
Ab7090, by Abcam (5 mentions)
Ripa buffer, by Solarbio (5 mentions)
Ab38449, by Abcam (5 mentions)
Ab2324, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Ab194583, by Abcam (4 mentions)
64 protocols using ab53154
1
Western Blot Analysis of Apoptosis and Autophagy
2
Phillyrin Modulates Oxidative Stress and Apoptosis in ARPE-19 Cells
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Human ARPE-19 cell line was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). Phillyrin (batch number Z17A8X34077, purity > 98%) was a product of the China Food and Drug Administration (Beijing, China). Dulbecco's Modified Eagle's Medium/nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin, and streptomycin solutions were purchased from Corning (NY, USA). Trypsin (0.05%) and phosphate buffered saline (PBS) were produced by Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT), ML385, N-acetylcysteine (NAC)m and H2O2 solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Fas (ab82419), cytochrome c (ab90529), cleaved caspase-3 (ab2302), cleaved caspase-9 (ab2324), NQO1 (ab80588), Keap1 (ab118285), Bcl-2 (ab185002), Nrf2 (ab62352), CDK2 (ab32147), cyclin A (ab33911), cyclin E (ab181591), Bax (ab53154), β-actin (ab8226), p21 (ab188224), Histone H3 (ab1791), COX IV (ab16056) p53 (ab241556), pro caspase-3, pro caspase-8, and pro caspase-9 were obtained from Abcam, Cambridge, (MA, USA). Antibodies against p-p53 (9286S), cleaved caspase-8 (9496S), cleaved PARP (5625S), and HO-1 (86806S) were purchased from Cell Signaling Technology, Beverly, (MA, USA).
3
Apelin, APJ, Bcl-2, and Bax Expression in Cardiac Tissue
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Western blotting was used to measure the apelin, APJ, Bcl-2, and Bax expression levels in the left ventricular tissues. The frozen tissues were weighed and homogenized in RIPA lysis buffer. Protein levels in the supernatant were determined using the Bradford method (21 (link)). Thereafter, 40 µg of protein was subjected to SDS-PAGE with 10% separation and transferred onto a nitrocellulose membrane for 3 h at 200 mA. The membranes were blocked with 1% bovine serum albumin for 1 h. Subsequently, primary antibodies for apelin (ab125213, dilution, 1:500; Abcam,
Cambridge, United Kingdom), APJ (ab214369, dilution, 1:500; Abcam, Cambridge, United Kingdom), Bcl-2 (ab59348, dilution, 1:500; Abcam, Cambridge, United Kingdom), and Bax (ab53154, dilution, 1:500; Abcam, Cambridge, United Kingdom) were added, followed by overnight incubation at 4°C. After washing twice with TBS, membranes were incubated with the respective secondary antibodies (dilution, 1:10,000; Santa Cruz, CA, USA). Finally, protein band intensities were quantified using a densitometer analysis system (Quantity One software, Bio-Rad, PA, USA).
Cambridge, United Kingdom), APJ (ab214369, dilution, 1:500; Abcam, Cambridge, United Kingdom), Bcl-2 (ab59348, dilution, 1:500; Abcam, Cambridge, United Kingdom), and Bax (ab53154, dilution, 1:500; Abcam, Cambridge, United Kingdom) were added, followed by overnight incubation at 4°C. After washing twice with TBS, membranes were incubated with the respective secondary antibodies (dilution, 1:10,000; Santa Cruz, CA, USA). Finally, protein band intensities were quantified using a densitometer analysis system (Quantity One software, Bio-Rad, PA, USA).
4
Western Blot Analysis of Protein Markers
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Total protein was isolated from treated HepG2 and HCCLM3 cells using RIPA lysis buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentrations across samples were measured using BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts (20 μg) of protein were separated by 10% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). After blocking with 5% skim milk, the membranes were incubated at 4°C overnight with primary antibodies against TIMP3 (ab39184; 1 : 1,000), TGF-β1 (ab9785; 1 : 200), p-smad3 (ab63403; 1 : 500), cleaved caspase-3 (ab2302), Bax (ab53154), Bcl-2 (ab196495), and β-actin (ab8227; 1 : 2,000) all from Abcam (Cambridge, MA, UK), followed by HRP-conjugated goat anti-rabbit IgG (ab7090; 1 : 10,000) at room temperature for 1 h. Signals were detected using an ECL kit (Millipore). β-actin served as an endogenous reference.
5
Protein Expression Analysis in Cell Lysates
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Tissue homogenates and cells were treated with RIPA lysates (Beyotime, China), and supernatants containing proteins were collected by centrifugation. Protein content was assessed by BCA detection kit (EMD Millipore). SDS-PAGE was performed with the same amount of protein samples in each lane, and then the isolated proteins were electrotransferred to PVDF membrane (Millipore). 5% skimmed milk powder was used for blocking the membranes, and then the corresponding primary antibody (eIF5A (1 : 1000, ab32443, Abcam); FANCD2 (1 : 1000, ab108928, Abcam); SLC7A11 (1 : 1000, ab216876, Abcam); HSPB1 (1 : 1000, ab109376, Abcam); Bax (1 : 1000, ab53154, Abcam); Bcl-2 (1 : 1000, ab32124, Abcam); cleaved caspase-3 (1 : 1000, ab32042, Abcam); cytochrome C (1 : 1000, ab133504, Abcam); β-actin (1 : 1000, ab8226, Abcam)) was applied overnight at 4°C. Following that, the membranes were treated for 2 h with the corresponding secondary antibody (Goat Anti-Rabbit IgG H&L (1 : 2000, ab6721, Abcam) and Rabbit Anti-Mouse IgG H&L (1 : 2000, ab6728, Abcam)). The transfer protein on membranes was developed with electrochemiluminescence (ECL, Thermo Fisher Scientific, USA)). Grayscale of the strips was assessed by ImageJ 1.48v software (NIH).
6
Protein Expression Analysis of Cancer Markers
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The total proteins were isolated by using lysis buffer containing a protease inhibitor cocktail (P8340; Sigma). The protein concentration in each sample was evaluated by using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). A 30 µg sample of total protein from each sample was separated by 10% SDS-PAGE, and the separated protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), which were then blocked with skimmed milk (5%) for 2 hrs at room temperature. Next, the membranes were incubated overnight at 4 °C with primary antibodies, followed by incubation with the secondary antibodies (HRP linked, Abcam, Cambridge, UK, ab205718, 1:2,000) at room temperature for 2 hrs. The blots were exposed by using ECL detection reagent (EMD Millipore) and analyzed by using ImageJ version 1.48 software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used in this study were anti-E-cadherin (Abcam, ab76319, 1:1,000), anti-MMP2 (Abcam, ab2462, 1:1,000), anti-MMP9 (Abcam, ab228402, 1:1,000), anti-N-cadherin (Abcam, ab76057, 1:1,000), anti-Bax (Abcam, ab53154, 1:1,000), anti-Caspase-3 (Abcam, ab214430, 1:1,000), and anti-GAPDH (Abcam, ab9485, 1:2,000).
7
Western Blot Analysis of Endothelial Cell Signaling
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HUVECs were lysed in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China), and protein concentration was measured by BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Next, membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight 4°C with the primary antibodies as follows: cathepsin C (Abcam, ab199109, 1:1000), p-p38 MAPK (Abcam, ab178867, 1:1000), p38 MAPK (Abcam, ab170099, 1:5000), p-NF-κB p65 (Abcam, ab76302, 1:1000), NF-κB p65 (Abcam, ab32536, 1:10,000), p-IκBα (Abcam, ab133462, 1:10,000), IκBα (Abcam, ab32518, 1:10,000), Bcl-2 (Abcam, ab196495, 1:2000), Bax (Abcam, ab53154, 1:1000), Birc5 (Abcam, ab76424, 1:5000), Cleaved PARP (Abcam, ab32064, 1:10,000), PARP (Abcam, ab191217, 1:1000) and GAPDH (Abcam, ab9485, 1:2500). On the second day, the membranes were incubated with the corresponding secondary antibody (Abcam, ab205718, 1:50,000) for 1.5 h at room temperature. Protein blots were visualized using electrochemiluminescence (ECL; Beyotime, Shanghai, China) method and analyzed by a Bio-Rad imaging system (Bio-Rad, CA, USA).
8
Protein Extraction and Western Blot Analysis
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Protein extraction was implemented utilizing RIPA Lysis Buffer (Beyotime). The concentrations of protein samples were examined with a BCA protein assay kit (Tiangen). After separation by 10% sodium dodecyl sulfonate‐polyacrylamide gel (SDS‐PAGE; Solarbio) electrophoresis, the protein sample (20 μg) was blotted onto polyvinylidene difluoride membrane (Corning) and then blocked with 5% non‐fat milk (Solarbio) for 1 h. Afterwards, the membranes were immunoblotted with primary antibodies against CHEK1 (1:10000, ab32531; Abcam), proliferating cell nuclear antigen (PCNA) (1:2000, ab152112; Abcam), Ki67 (1:1000, ab243878; Abcam), matrix metalloproteinase 9 (MMP9) (1:1000, ab38898; Abcam), Bcl‐2‐associated X protein (Bax, 1:1000, ab53154; Abcam) and GAPDH (1:10000, ab181602; Abcam) overnight at 4°C followed by interaction with Goat Anti‐Rabbit IgG H&L (HRP) secondary antibody (1:10000, ab205718; Abcam) for 2 h at indoor temperature. Finally, the intensity of protein bands was assessed utilizing BeyoECL Plus ECL Kit (Beyotime).
9
Western Blot Analysis of Cell Signaling
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The protein sample was added to the corresponding proportion of SDS gel loading buffer and boiled for 5 min. After SDS-PAGE electrophoresis and transferred membrane, the membrane was blocked 5% skim milk in PBST buffer, at room temperature for 1 hour and washed with PBST for 3 times. Then ERK1/2, p-ERK, p38, p-p38, JNK, p-JNK, and Bcl-2 (ab321124, Abcam), Bax (ab53154, Abcam), and Caspase3 (ab44976, Abcam) (1:500) primary antibody were added, and the membranes were incubated overnight at 4°C, washed 3 times with PBST, and incubated in the corresponding secondary antibody labeled with horse. Then the membranes were added by radish peroxidase and incubated for 1 hour at room temperature. The membrane was washed 3 times and ECL developed. The gel imaging system was photographed, and the image J software was used to analyze the gray value.
10
Immunohistochemical Analysis of Heart and Pancreas
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Paraffin-embedded sections of the heart and pancreas were deparaffinized in xylene and then processed for immunohistochemical staining using the labeled streptavidin-biotin immunoperoxidase technique [30 (link)]. The mouse monoclonal anti-p53 antibody (DO-1: sc-126) was obtained from Santa Cruz, CA, USA; diluted 1:50). The rabbit polyclonal primary antibodies for caspase-3 (ab13847), Bax (ab53154) and Bcl-2 (ab59348) were obtained from Abcam, Cambridge, MA, USA. The dilution was 1:500 for caspase-3 and 1:50 for Bax and Bcl-2. The sections incubated with the primary antibodies overnight at 4 °C. The labeling index was assessed as described previously [31 (link)] using Image J software [32 (link)].
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