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 bcl 2
Manufactured by ABclonal
Sourced in China, United States
Anti-Bcl-2 is a laboratory reagent that targets the Bcl-2 protein. Bcl-2 is a key regulator of apoptosis, or programmed cell death. Anti-Bcl-2 is used in research applications to study the role of Bcl-2 in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti bax, by ABclonal (10 mentions)
Anti caspase 3, by ABclonal (5 mentions)
Anti akt, by ABclonal (5 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Anti β actin, by ABclonal (4 mentions)
Anti p akt anti alix, by ABclonal (4 mentions)
Biotrace, by Bio-Rad (4 mentions)
Anti cd81, by ABclonal (4 mentions)
Anti calnexin, by Bioworld Technology (4 mentions)
Anti tsg101, by ABclonal (4 mentions)
Anti cd63, by ABclonal (4 mentions)
Anti hmgb1, by ABclonal (4 mentions)
Anti gapdh, by ABclonal (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti gapdh, by Proteintech (2 mentions)
Anti caspase 1, by ABclonal (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti gsk3β, by ABclonal (2 mentions)
Bca assay, by Beyotime (1 mentions)
Anti β actin ac006, by ABclonal (1 mentions)
28 protocols using anti bcl 2
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Signaling Proteins
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Proteins were subjected to SDS-PAGE on polyacrylamide gels (8–10%) and transferred onto a PVDF membrane. After blocking with 5% non-fat milk in TBS containing 0.1% Tween-20, the membrane was incubated at 4 °C overnight with one of the following primary antibodies: anti-p-NF-κB P65, anti-NF-κB P65, anti-p-mTOR, anti-p-ERK, anti-ERK, anti-PDCD4, anti-GNA12 (Affinity, USA); anti-mTOR, anti-GAPDH, anti-TSG101, anti-CD63, anti-CD81, anti-Bax (Proteintech, USA); anti-Bcl-2 (Abclonal, China). Subsequently, the peroxidase-conjugated AffiniPure goat anti-rabbit or mouse IgG (Proteintech, USA) was added. Bound antibody was visualized via ECL plus TM Western blotting system detection kit (Amersham, USA).
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
Western Blotting Analysis of Apoptosis and Autophagy Markers
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The total protein was extracted with RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitors and phosphatase inhibitors. Then, the total protein was quantified with the BCA protein analysis kit (Solarbio, Beijing, China). The protein was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membrane. Block the membrane in TBST buffer with 5% milk for 90 minutes, and incubate at 4°C overnight with anti-TP63 (Santa cruz, dilution 1:200), anti-P62/SQSTM1 (Abnova, dilution 1:1000), anti-Beclin1 (ABclonal, dilution 1:1000), anti-Bax (BOSTER, dilution 1:1000), anti-Bcl-2 (ABclonal, dilution 1:1000) and anti-GAPDH (ABclonal, dilution 1:40000) primary antibodies. Next, the secondary antibody (EarthOx, dilution 1:40000) labeled with horseradish peroxidase (HRP) was used for 2 hours at room temperature. A chemiluminescence imaging system (Amersham Imager 680, GE, USA) was used to detect the band intensity. The results were normalized to GAPDH, and Image J (National Institute of Mental Health, Bethesda, USA) was applied for band density analysis.
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
Antibody-based Protein Analysis in Cells
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Specific antibodies used were: anti-ΔNp63α, anti-CB1 and anti-cIAP2/BIRC3 (GeneTex, Irvine, CA, USA); anti-AKT and anti-Phospho-AKT-Ser473 (Cell Signaling Technology, Danvers, MA, USA); anti-β-catenin (Thermo Scientific, Waltham, MA, USA); anti-P53 (Cell Signaling Technology, Danvers, MA, USA); anti-Bcl-2 (Abclonal, Woburn, MA, USA) and anti-Ki67 (Santa Cruz Biotechnology INC., Santa Cruz, CA, USA).
8
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.
9
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).
10
Protein Expression Analysis in Cells and Tumor Tissues
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Cells and tumor tissues were lysed with RIPA lysis buffer. Equal amounts of proteins were separated by SDS-PAGE electrophoresis and then transferred onto PVDF membranes (Millipore). After blocking with 5% milk for 2 h, the blots were probed with the anti-AGK (Abcam, ab137616), anti-BCL-2 (Abclonal, A19693), anti-PTEN (CST, #9188), anti-pPTEN (CST, #9551), anti-AKT (CST, #4691), anti-pAKT(T308) (CST, #2965), anti-pAKT (S473) (CST, #4060), anti-FOXO1 (CST, #2880), anti-pFOXO1 (CST, #9464), anti-β-Actin (CST, #4970) and anti-GAPDH (Servicebio, GB11002) antibodies overnight at 4 °C. Then, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody for 1.5 h, followed by ECL detection (GE Healthcare).
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