The total protein concentration was determined by BCA assay (Beyotime, China). And the protein concentrations were adjusted to 1μg/μL. Cell lysates were boiled at 100°C for 5 min, separated on 12% SDS–PAGE and transferred to 0.22 μm PVDF membrane. Membranes were blocked with 5% non-fat milk at room temperature for 1.5 h. Then, the membranes were incubated with the primary antibodies overnight at 4 °C, followed by incubation with the secondary antibodies. Immunoblots were visualized using ECL and analyzed using Image J software. The following antibodies (Abclonal, China) were used: anti-β-actin (AC006), anti-caspase3(A19654), anti-PARP (A11010), anti-Bax (A12009), anti-Bcl2(A19693) and HRP Goat Anti-Rabbit IgG (H+L) (AS014).
Anti bax
Manufactured by ABclonal
Sourced in China, United States
Anti-Bax is a laboratory reagent that specifically detects the Bax protein, a key regulator of apoptosis (programmed cell death). It is designed for use in various cell and molecular biology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2, by ABclonal (10 mentions)
Anti caspase 3, by ABclonal (2 mentions)
Anti β actin, by ABclonal (2 mentions)
Anti β actin, by Proteintech (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti β actin ac006, by ABclonal (1 mentions)
Bca assay, by Beyotime (1 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
A11136, by ABclonal (1 mentions)
A19607, by ABclonal (1 mentions)
A19083, by ABclonal (1 mentions)
A11355, by ABclonal (1 mentions)
Ap0115, by ABclonal (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
A19056, by ABclonal (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
Anti tlr4, by Bioss Antibodies (1 mentions)
Anti caspase 3, by Wuhan Servicebio Technology (1 mentions)
Anti myd88, by Bioss Antibodies (1 mentions)
15 protocols using anti bax
1
Protein Expression Analysis by Western Blot
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA buffer (R0010, Solarbio, Beijing, China). Protein extracts were transferred to PVDF membranes, and then incubated using appropriate antibodies. An ECL imaging system (Tanon-5200, Shanghai, China) was used to detect ECL signals. The primary antibodies used were: anti-LTB4R (ERP7113, 1:1000, Abcam, Carlsbard, CA, USA); anti-GAPDH (A19056, 1:1000, ABclonal, Hubei, China), anti-CDK2 (A0094, 1:1000, ABclonal), anti-CDK4 (A11136, 1:1000, ABclonal), anti-cyclin D1 (A19038, 1:1000, ABclonal), anti-Bcl-2 (A19693, 1:1000, ABclonal), anti-Bax (A19684, 1:1000, ABclonal), anti-caspase3 (9662, 1:1000, Cell Signaling Technology), anti-cleaved-caspase3 (Asp175, 1:1000, Cell Signaling Technology), anti-vimentin (A19607, 1:1000, ABclonal), anti-NCA (A19083, 1:1000, ABclonal), anti-ECA (A18135, 1:1000, ABclonal), anti-AKT (A17909, 1:1000, ABclonal), anti-P-AKT (AP0637, 1:1000, ABclonal), anti-mTOR (A11355, 1:1000, ABclonal), and anti-P-mTOR (AP0115, 1:1000, ABclonal). The second antibody was Anti-Rabbit-IgG(H+L)-HRP (AS030, 1:10,000, ABclonal).
3
Immunohistochemical Analysis of Apoptosis Markers
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Tissues were embedded using paraffin and cut into 4-μm-thick sections (n = 3 animals per group, 6 sections per animal). Sections were deparaffinized and rehydrated, then placed in a box filled with citric acid antigenic repair buffer (pH 6.0) in a microwave oven for antigen retrieval. Later, they were incubated with 3% H2O2 for 10 min at room temperature. After blocking with 5% BSA for 30 min, the tissue sections were incubated with primary antibody anti-Caspase1 (ABclonal, Wuhan, China; Catalog: A0964, diluted at 1:200), anti-Caspase3 (Servicebio, Wuhan, China; Catalog: GB11009-1, diluted at 1:200), anti-TLR4 (Bioss, Beijing, China; Catalogue: bs-20594R, diluted at 1:200), anti-MYD88 (Bioss, Beijing, China; Catalogue: bs-1047R, diluted at 1:200), anti-BAX (ABclonal, Wuhan, China; Catalog: A0207, diluted at 1:500), and anti-BCL-2 (ABclonal, Wuhan, China; Catalog: A0208, diluted at 1:500). The secondary antibody was incubated at room temperature. After PBS washing, the sections were monitored and captured under a microscope.
4
Protein Expression Profiling in Cells and Tissues
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Cells and heart tissues were lysed in lysis buffer containing protease inhibitors. Later on, the proteins were separated via 10% SDS-PAGE and transferred to a PVDF membrane. Next, the membranes were blocked in 5% milk for 2 h, before their incubation with the anti-Yap (Affinity Biosciences, cat. no. DF3182; 1:1000; China), anti-Ki67 (Affinity Biosciences, cat. no. AF0198; 1:2000; China), anti-Bax (Abclonal, cat. no. A19684; 1:2000; China), anti-Bcl2 (ABclonal, cat. no. A19693; 1:1000; China), and anti-GAPDH (Affinity Biosciences, cat. no. AF7021; 1:3000; China) at 4 °C overnight. The membranes were washed with TBST and incubated with HRP-conjugated anti-rabbit IgG (Affinity Biosciences, cat.no. S0001; 1:3000; China) for 2 h. The proteins were visualized by enhanced chemical luminescence reagents. The band intensity was analyzed with the software of Image J.
5
Mitochondrial Dynamics and Cell Signaling
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NaHS was purchased from Sigma-Aldrich (St Louis, MO, USA), and the cigarettes were purchased from Guangdong Tobacco Industry Co., Ltd. (Guangzhou, China). SRT1720 and EX 527 were purchased from Selleck Chemicals (Houston, TX, USA). The TRIzol Reagent was purchased from Ambion (Life Technologies, CA, USA). The PrimeScript RT reagent Kit with gDNA Eraser was from Takara Bio Inc. (Takara, Shiga, Japan), and the SsoFast EvaGreen Supermix was obtained from Bio-Rad Laboratories, Inc. (CA, USA). The primary and second antibodies described in this study include: anti-Bcl-2 and anti-β-actin polyclonal antibodies were purchased from Proteintech (Chicago, IL, USA); anti-MFN1, anti-SIRT1, anti-FOXO3 and anti-Bax antibodies were purchased from Abclonal (Wuhan, China); anti-p21 and anti-p53 antibodies were purchased from Cell Signaling Technology (CA, USA); anti-FIS1 antibodies, and the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit/Mouse IgG (H+L) were purchased from Abcam Biotechnology (Cambridge, MA, USA). The poly-vinylidene fluoride (PVDF) membranes were from Millipore Corporation (Billerica, MA, USA). ECL-Plus detection kit probed was purchased from Tanon Science and Technology Co., Ltd. (Shanghai, China). Other reagents were all purchased from GBCBIO Technologies Inc. (Guangzhou, China) unless otherwise indicated.
6
Protein Expression Analysis in Cells
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Cells were washed twice in cold PBS and total protein was extracted using RIPA lysate (cat. no. P0013C; Beyotime Institute of Biotechnology) containing protease inhibitor (Merck KGaA). Total protein was quantified using the bicinchoninic acid protein assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology). Proteins (40 µg) were separated via 10% SDS-PAGE and transferred onto PVDF membranes, which were blocked in 5% skim milk at room temperature for 1 h. The membranes were incubated the following primary antibodies at 4°C: Anti-FoxM1 (cat. no. A2493; ABclonal Biotech Co., Ltd.), anti-Bax (cat. no. A0207; ABclonal Biotech Co., Ltd.), anti-Bcl-2 (cat. no. 2870; Cell Signaling Technology, Inc.) and anti-tubulin (cat. no. 2144; Cell Signaling Technology). Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 1 h. Protein bands were visualized using ECL plus reagent (Thermo Fisher Scientific, Inc.) and a Chemo XRS+ luminometer (Bio-Rad Laboratories). Protein expression levels were quantified using Quantity One software (Bio-Rad Laboratories) with tubulin as the loading control.
7
Apoptosis Pathway Regulatory Genes Analysis
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The levels of Bcl-2, Bax, Caspase3, and PARP1, regulatory genes involved in the intracellular Ca2+ homeostasis, and apoptosis pathways, were determined by western blot analysis. Cells were harvested and lysed for western blot analysis. Lysates were prepared as previously described (Zhang et al., 2019 (link)). Membranes were incubated with the following primary antibodies: anti Bcl-2 (1:800, ABclonal, United States), anti Bax (1:800, ABclonal, United States), anti Caspase3 (1:1,000, ABclonal, United States), anti PARP1 (1:1,000, ABclonal, United States). HRP-conjugated secondary Abs were applied, and a supersensitive ECL Chemiluminescence Kit (Beyotime, China) was used to detect proteins. anti-β-actin (1:1,600, ABclonal, United States) was used as a loading control.
8
Rhein Modulates AMPK Signaling and Apoptosis
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Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is an anthraquinone compound isolated from rhubarb. Rhein [> 98% high-performance liquid chromatography (HPLC) purity] was purchased from Maclaurin (Shanghai, China). Antibodies against phosphor-AMPKα, AMPKαand GAPDH were purchased from Cell Signaling Technology (Boston, USA). The Anti-atrial natriuretic peptide (ANP), Anti-brain natriuretic peptide (BNP), Anti-FGF23, Anti-BAX and Anti-BCL-2 antibodies were from Abclonal (Wuhan, China).
9
Western Blot Analysis of Protein Targets
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According to the molecular weight of the target protein, cells or tissue samples containing the same amount of protein were loaded onto appropriate concentration SDS-PAGE for electrophoretic separation, and subsequently transferred to the PVDF membrane (Millipore, Billerica, MA, USA), which was then blocked in the blocking buffer (Epizyme, Shanghai, China) for 1 h at room temperature, followed by incubation overnight at 4 °C with the following primary antibodies: anti-β-actin (AC038, Abclonal, Wuhan, China), anti-STAT1 (AF6300, Affinity Biosciences, Changzhou, China), anti-phospho-STAT1 (AF3300, Affinity), anti-STAT3 (AF6294, Affinity), anti-phospho-STAT3 (AF3293, Affinity), anti-Bcl2 (A0208, Abclonal), anti-Cleaved-Caspase3 (9664S, Cell Signaling Technology), anti-GFAP (GB12096, Servicebio), anti-MAP2 (A0453, Abclonal), anti-Bax (A19654, Abclonal), anti-iNOS (13120S, Cell Signaling Technology), and anti-Arg1 (93668S, Cell Signaling Technology). On the following day, the membrane was incubated with relevant HRP-conjugated secondary antibody (S0001, S0002, Affinity) for 2 h, and visualized using ECL reagent (SD6032, Simuwu, Shanghai, China). The protein band images were obtained on the Chemiluminescent Imaging System (Tanon, 5200, China). All experiments were repeated three times. The density of protein bands was semi quantitatively analyzed by ImageJ software.
10
Western Blot Analysis of Apoptosis Markers
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Total proteins were extracted from mouse heart tissues or NRCMs with radio immunoprecipitation assay (RIPA, KeyGen BioTECH, China) lysis buffer. The protein concentration was quantified by TaKaRa BCA Protein Assay Kit (TaKaRa, Japan). Equal amounts of protein (20–30 μg) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride membrane, and blocked with 5% BSA (KeyGen BioTECH, China) in Tris-buffered saline Tween (TBST, 0.1% Tween 20) for 2 h at room temperature. The membrane was then incubated with anti-cleaved-Caspase3 and anti-Caspase3 (1:1000, Abclonal, Wuhan, China, A11040), anti-Bax (1:1000, Abclonal, Wuhan, China, A12009), anti-Bcl-2 (1:1000, Abclonal, Wuhan, China, A19693), anti-BTG2 (1:1000, Abcam, Shanghai, China, ab197362), anti-GAPDH (1:1000, Bioworld, Nanjing, China, AP0063) and anti-β-actin (1:10000, Abclonal, Wuhan, China, AC004) at 4 °C overnight. Then, the anti-rabbit (1:10000, Jackson, USA, 111-035-003) or anti-mouse (1:10000, Bioworld, Nanjing, China, BS12478) second antibody was added and the membrane was incubated with the second antibody for 2 h at room temperature. The membrane was visualized in High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China). Image J software was used for gray scale analysis.
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