Protein concentrations were determined with a BCA protein assay kit (Beyotime, P0012i). Twenty to thirty micrograms of total protein was separated by 10% SDS-PAGE (EpiZyme, pg112) and then transferred onto nitrocellulose membranes (Pall, 66485). Nonspecific sites were blocked with 5% nonfat milk in phosphate-buffered saline (with 0.1% Tween-20) at room temperature. Next, the blots were incubated overnight at 4 ℃ with the following primary antibodies: anti-Hbα (Santa Cruz, sc-514378), anti-Hbβ (Santa Cruz, sc-21757), anti-CD31 (Affinity, AF6191), anti-glutathione peroxidase 4 (GPX4) (Affinity, DF6701), anti-heme oxygenase-1 (HO-1) (Affinity, AF5393), anti-Bcl2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-Notch1 (Cell Signaling Technology, #3608), anti-Jag1 (Cell Signaling Technology, #70109), anti-Hes1 (Abcam, ab108937), anti-Hey1 (Abcam, ab154077), and anti-β-actin (Affinity, AF7018). The next day, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity, s0001) and visualized with New Super ECL assay kit (KeyGen BioTECH, KGP1128).
Anti bax
Manufactured by Immunoway
Sourced in United States, Japan
Anti-Bax is a laboratory reagent used to detect and quantify the expression of the Bax protein, a key regulator of apoptosis (programmed cell death). It is designed for use in various research applications, such as Western blotting, immunohistochemistry, and flow cytometry, where the analysis of Bax expression levels is of interest.
Automatically generated - may contain errors
Lab products found in correlation
Anti bcl 2, by Immunoway (5 mentions)
Anti bcl 2, by Bioss Antibodies (2 mentions)
Anti actin, by Abcam (2 mentions)
Anti caspase 3, by Abcam (2 mentions)
Anti nrf2, by Bioss Antibodies (2 mentions)
Anti ho 1, by Bioss Antibodies (2 mentions)
Anti hes1, by Abcam (1 mentions)
Anti jag1, by Cell Signaling Technology (1 mentions)
Anti hey1, by Abcam (1 mentions)
Anti cd31, by Affinity Biosciences (1 mentions)
Anti notch1, by Cell Signaling Technology (1 mentions)
New super ecl assay kit, by Keygen Biotech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti β actin, by Affinity Biosciences (1 mentions)
Anti bad, by Abcam (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Kgpbca, by Keygen Biotech (1 mentions)
Kgp250, by Keygen Biotech (1 mentions)
Anti yap taz, by Cell Signaling Technology (1 mentions)
Cell lysis buffer for western and ip, by Beyotime (1 mentions)
10 protocols using anti bax
1
Western Blot Analysis of Protein Markers
2
Protein Expression Analysis after Drug Treatment
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The cells were collected after drug intervention. Whole cell lysates were obtained by gentle lysis (Cat. No. KGP250, KeyGEN). Protein quantitation was performed by the BCA method (Cat. No. KGPBCA, KeyGEN). The protein was separated by SDS-PAGE, and the protein was transferred to PVDF membranes by wet-transfer method. Thereafter, the membrane was incubated with 50 mL/L skim milk powder at room temperature for 1 h. Later on, the membranes were incubated with the primary antibodies, including anti-MRPL35 (Cat. No. YT5669, Immunoway; 1:2000), anti-TP53 (Cat. No. YM3052, Immunoway; 1:2000), anti-BCL2L1 (Cat. No. YT0477, Immunoway; 1:2000), anti-BAX (Cat. No. YM3619, Immunoway; 1:500), anti-COPS5 (Cat. No. ab124720, Abcam; 1:2000), anti-BAD (Cat. No. 9292, Cell Signaling Technology; 1:1000), and anti-β-tubulin (Cat. No. YM3030, Immunoway; 1:10000). Then, the membranes were washed with TBST and incubated with anti-mouse/rabbit IgG antibody (Cat. No. S001 S004, TDYBio; 1:10000) at 37 ℃ for 1 h. After washing, the proteins were detected with an ECL detection system. ImageJ software was used to measure the band intensity.
3
Huaier Modulates Cellular Signaling
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Cells were treated with a series concentrations of Huaier (0, 5, 10 and 15mg/ml) for 24h, and then lysed by cell lysis buffer for Western and IP (Beyotime, P0013). Protein concentration was detected with BCA Protein Assay Kit (KeyGen BioTECH, KGP902). After that, 20µg proteins were separated by 10% or 12% SDS-PAGE and transferred onto Pure Nitrocellulose Blotting Membrane (Pall Corporation, P/N 66485). Then, the blots were blocked for nonspecific binding with 5% non-fat milk in PBST (PBS, Tween-20, pH7.4) at room temperature for 1h. Next, the blots were incubated overnight at 4℃ with 5% non-fat milk containing primary antibodies which are listed as follows: anti-PCNA (ImmunoWay, YM3031), anti-Ki-67 (ImmunoWay, YT2467), anti-β-actin (ImmunoWay, YM3028), anti-Bcl-2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-E-cadherin (ImmunoWay, YT1454), anti-N-cadherin (ImmunoWay, YT2988), anti-YAP1 (abcam, ab52771), anti-p-YAP1 (ab76252), anti-CyclinD1 (abcam, ab134175), anti-Cleaved Caspase Substrate Motif (Cell Signaling, #8698) and anti-YAP/TAZ (Cell Signaling, #8418). After that, incubating blots with secondary antibodies conjugated with HRP (Cell Signaling, #7074 or #7076) for 1h at room temperature. Finally, after washing three times with PBST, the blots were visualized by New Super ECL Assay (KeyGen BioTECH, KGP1128).
4
Western Blot Analysis of Apoptosis Markers
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Cultured or transfected cells were lysed in RIPA buffer with 1% PMSF. Protein was loaded onto a SDS‐PAGE minigel and transferred onto a PVDF membrane. After being probed with primary antibody (anti‐Mcl‐1, anti‐Bcl‐2, anti‐Bax, anti‐cytochrome c, anti‐cleaved caspase 9, anti‐cleaved caspase 3, and anti‐cleaved PARP from ImmunoWay Biotechnology Company, Plano, TX, USA; anti‐Bcl‐2 from ImmunoWay Biotechnology Company, Plano, TX, USA; and anti‐β‐actin from Abcam, Cambridge, UK) at 4°C overnight, the blots were subsequently incubated with secondary antibody (1:5000, Auragene, Changsha, China). Signals were visualized using ECL Substrates (Millipore, MA, USA). β‐actin was used as an endogenous protein for normalization. The results were assessed by IPP6.0 software.
5
Western Blot Analysis of Protein Expression
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Total protein was extracted from cell lysates using radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China). Equal amounts of total protein samples were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred from the gel to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk or bovine serum albumin, and then immunoblotted using the following primary antibodies: rabbit anti-PGAM1 (1:1000; Abcam), rabbit polyclonal anti-cleaved caspase-3 (1:500, #9664; Cell Signaling Technology, Danvers, MA, USA); and rabbit polyclonal anti-Bcl-2, anti-Bax, anti-matrix metallopeptidase (MMP)-2, and anti-MMP-9 (all 1:500; all Immunoway, Plano, TX, USA). Anti-β-actin staining (1:1000; Bioworld Technology, Louis Park, MN, USA) was used as an internal control. Finally, the membranes were incubated with the appropriate secondary antibodies (1:5000; Boster Ltd., Wuhan, China). Signals were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology, Rockford, IL, USA) in accordance with the manufacturer's instructions.
6
Protein Expression Analysis by Western Blot
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Whole protein lysates were extracted from the cells with RIPA lysis buffer (Solarbio, China) added PMSF and quantified by BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of protein were fractionated on 10% SDS-PAGE. Western blotting was performed as previously described. The primary antibodies used were as follows: mouse anti-α-F1-ATPase (Abcom, Cambridge, MA), rabbit monoclonal anti-Bax (ImmunoWay, USA), anti-Bcl2 (GeneTax, USA), anti-caspase3 (Signaling Technology, USA) and anti-β-actin (Signaling Technology, USA) antibodies (1:500). HRP-linked anti-mouse or anti-rabbit IgG antibodies (Zsjqbio, China) were used as secondary antibodies (1:10000). The antigens were visualized by the electrochemiluminescence (Millipore, Darmstadt, Germany). The results were normalized by β-actin levels.
7
Evaluating Antioxidant Protein Expressions in Chicken Kidneys
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Expressions of Nrf2, HO-1, CAT, MnSOD, Capase3, Bax, and Bcl-2 proteins in the kidneys of chickens were determined by western blot. The Protein Extraction Kit (Beyotime, Wuhan, China) was used to extract total proteins from the tissues. The BCA Protein Assay Kit (Solarbio, Beijing, China) was used to measure total protein content. The following antibodies were used to detect their respective proteins: anti-Caspase3 (1 : 1000, Abcam, Tokyo, Japan), anti-Bax (Immunoway, Suzhou, China), anti-HO-1, anti-Bcl-2, anti-Nrf2 (1 : 1000, Bioss, Beijing, China), anti-MnSOD (1 : 6000, Enzo Clinical Labs, Farmingdale, NY, USA), anti-CAT (1 : 700, Biorbyt, Cambridge, UK), anti-Actin (1 : 10000, Abcam, Tokyo, Japan). An HRP-labeled goat anti-rabbit IgG (1 : 10000, Jackson Immuno Research Labs, West Grove, PA, USA) was used as the secondary antibody. Relative band intensities were detected on a DNR Bio Imaging system by using the NcmECL Ultra method (Ncmbio, Suzhou, China). Relative intensities of these bands were normalized according to the β-actin.
8
Protein Extraction and Western Blotting
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For protein extraction, cells were lysed in ice-cold immunoprecipitation (IP) buffer containing phosphatase inhibitors, proteinase inhibitors, and phenylmethanesulfonyl fluoride (PMSF). Whole-cell lysates were centrifuged at 10,000 × g for five minutes at 4°C, and the protein concentrations were determined using a BCA protein assay kit. The protein extracts were separated into 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight at 4°C with these primary antibodies: anti-β-actin (60008–1, Proteintech, Wuhan, China), anti-GFP (Protein Tech Group, Inc., Chicago, US), anti-IKKβ (ab178870, Abcam, Cambridge, UK), anti-p-Erk1/2 (#4370, Cell Signaling Technology), anti-Bcl-2 (YM3041, Immunoway), and anti-bax (YT0458, Immunoway). Membranes were incubated for one hour at room temperature with IRDyeTM700DX anti-mouse IgG, IRDyeTM800DX anti-rabbit IgG, or IRDyeTM800DX anti-goat IgG (Rockland, Maine, US) conjugated secondary antibody (1:3000). The bands of interest were detected using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, US).
9
Oxidative Stress Mitigation in Cells
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OTA (purity > 98%) was provided by Pribolab (Immunos, Singapore); Se-Y (contained Se 2000 mg/kg) was obtained from Angel Yeast (Beijing, China) and was dissolved in autoclaved saline eluent. Kits for analyzing ALT, AST, MDA, GSH-Px, SOD and T-AOC contents were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Total tissue RNA extraction kit, cDNA synthesis kit and qRT-PCR kit were acquired from Vazyme (Nanjing, China). Anti-Caspase3 (1:900, Abcam, Tokyo, Japan), anti-Bax (1:900, Immunoway, Suzhou, China), anti-HO-1, anti-Bcl-2, anti-Nrf2 (1:800, Bioss, Beijing, China), anti-MnSOD (1:6000, Enzo, Farmingdale, NY, USA), anti-actin (1:10,000, Abcam, Tokyo, Japan) and the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:12,000, Jackson Immuno, PA, USA).
10
Protein Expression Analysis of Ovarian Tissue
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The ground ovarian tissue samples were lysed using RIPA buffer (Biotechwell, Shanghai, China). Total proteins were isolated by centrifuging the lysates at 12,000 rpm for 5 min. Protein concentrations were measured using a bicinchoninic acid kit (Biotechwell). The proteins were separated using gel electrophoresis and then transferred to a polyvinylidene uoride membrane. The membrane was blocked using 5% BSA for 2 h at room temperature, followed by an overnight incubation with anti-TLR (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-Bcl-2 (1:1000; A nity, Scoresby, Victoria, Australia), anti-collagen type I alpha 1 chain (Col1A1; 1:1000; A nity), anti-α-SMA (1:1500; Servicebi), anti-BAX (1:1500; ImmunoWay Biotechnology, Plano, TX, USA), anti-P-P65 (1:1000; ImmunoWay Biotechnology), anti-MyD88 (1:1000; ImmunoWay Biotechnolog), anti-P65 (1:1000; A nity), or anti-GAPDH (1:2000; Biotechwell) at 4°C. After a 2-h incubation with a secondary antibody (1:2000; Jackson Immuno, West Grove, PA, USA) at room temperature, the protein bands were developed using an enhanced chemiluminescence kit (Biotechwell).
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