Whole cell extracts were obtained by lysing cells in a buffer containing 25 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 1% SDS, plus protease and phosphatase inhibitors. The protein concentration was determined by the BCA protein assay. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% non fat milk, washed with 0.1% Tween/PBS and incubated overnight with a specific primary antibody. Membranes were washed and probed with the appropriate secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). The antibodies against Caspase 3 (#96615) and Caspase 7(#9492) were obtained from Cell Signaling, α-tubulin antibody (#T8203) was obtained from Sigma-Aldrich and Bax (Sc 493) was obtained from Santa Cruz Biotechnology.
Bax sc 493
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Bax (sc-493) is a lab equipment product offered by Santa Cruz Biotechnology. It is a protein that plays a key role in the regulation of apoptosis, or programmed cell death. The core function of Bax is to induce the release of cytochrome c from the mitochondria, which is a crucial step in the apoptotic process.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Bio-Rad (2 mentions)
Horseradish peroxidase, by Cytiva (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Caspase 7, by Cell Signaling Technology (1 mentions)
α tubulin antibody, by Merck Group (1 mentions)
β actin ab8226, by Abcam (1 mentions)
Nrf2 sc 13032, by Santa Cruz Biotechnology (1 mentions)
Bcl 2 sc 7382, by Santa Cruz Biotechnology (1 mentions)
Puma 55120 1 ap, by Proteintech (1 mentions)
Sxbp1, by Cell Signaling Technology (1 mentions)
Ero1α, by Cell Signaling Technology (1 mentions)
Catf6, by Abcam (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
β actin 60008 1 ig, by Proteintech (1 mentions)
Ab100768, by Abcam (1 mentions)
Cleaved caspase 3 sc 7148, by Santa Cruz Biotechnology (1 mentions)
Lk2001 lk2003, by Sungene Biotech (1 mentions)
Protease inhibitor, by Solarbio (1 mentions)
6 protocols using bax sc 493
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of Antioxidant and Apoptosis Markers
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Western blot analysis was conducted as previously reported [9 ]. In this study, two aortas from the same group were combined and homogenized to extract protein for the western blot assay [24 (link)25 (link)]. The protein content was quantified using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Protein expression was visualized by enhanced chemiluminescence and detected with CAS-400 apparatus (Core Bio, Seoul, Korea). The band densities were calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to the expression of β-actin. The antibodies used in this study; nuclear factor (erythroid-derived 2)-like 2 (Nrf2, sc-13032), superoxide dismutase (SOD, sc-11407), catalase (CAT, sc-34285), glutathione peroxidase (GSHPx, sc-133160), GRP78 (sc-1050), phospho-PERK (p-PERK, sc-32577), X-box binding protein 1 (XBP1, sc-7160), Bcl-2 (sc-7382), and Bax (sc-493) were purchased from Santa Cruz Biotech. (Santa Cruz, CA, USA). Phospho-JNK (p-JNK, MA5-14943) was from Thermo Scientific (Waltham, MA, USA). Phospho-eukaryotic initiation factor 2 subunit alpha (p-eIF2α, ab32157), caspase-3 (ab32351), caspase-9 (ab63488), cellular inhibitor of apoptosis protein (cIAP, ab25939-100), and β-actin (ab8226) were from Abcam Inc. (Cambridge, UK).
3
Antibody Validation for ER Stress Signaling
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Antibodies for KDEL receptor (sc-33806, 1:50 dilution for immunostaining), Bax (sc-493, 1:500 dilution for western blot), and Bcl2 (sc-7382, 1:1000 dilution for western blot) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for CHOP (#2895, 1:1000 dilution for western blot), cleaved caspase-3 (#9662, 1:1000 dilution for western blot), Ero1α (#3264, 1:1000 dilution for western blot), sXBP1 (#12782, 1:1000 dilution for western blot), and cleaved PARP (#9542, 1:1000 dilution for western blot) were from Cell Signaling Technology (Boston, MA, USA). Antibodies for BiP (ab21685, 1:500 dilution for western blot), cATF6 (ab11909, 1:1000 dilution for western blot), and GAPDH (ab9484, 1:1000 dilution for western blot) were from Abcam (Cambridge, MA, USA). Antibodies for AGGF1 (11889-1-AP, 1:1000 dilution for western blot), ATF4 (10835-1-AP, 1:500 dilution for western blot), β-actin (60008-1-Ig, 1:1000 dilution for western blot), DR5 (15497-1-AP, 1:500 dilution for western blot), and Puma (55120-1-AP, 1:500 dilution for western blot) were from Proteintech (Wuhan, Hubei, China). An antibody for p-PERK (DF7576, 1:1000 dilution for western blot) was from Affinity Biosciences (Cincinnati, OH, USA), and an antibody for p-eIF2α (1:200 dilution for western blot) was from Bioss (Beijing, China).
4
Testicular Protein Expression Analysis
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All testicular tissue proteins were extracted with radioimmunoprecipitation assay lysis buffer (P0013B; Beyotime Institute of Biotechnology, Haimen, China) then quantified using a bicinchoninic acid assay. Briefly, 40 µg/lane protein samples were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked at 37°C for 2 h with 5% non-fat milk in TBST buffer and then incubated with the following primary antibodies at 4°C overnight: Bax (sc-493; 1:500; Santa Cruz Biotechnology, Inc.), cleaved-caspase-3 (sc7148; 1:1,000; Santa Cruz Biotechnology, Inc.), tumor necrosis factor (TNF)-α (ab6671; 1:500; Abcam, Cambridge, UK) and interleukin (IL)-1β (ab100768; 1:200; Abcam). Following three washes with TBST buffer, membranes were incubated with secondary goat anti-rabbit antibodies conjugated with horseradish peroxidase (LK2001/LK2003; 1:100; Sungene Biotech, Co., Ltd., Tianjin, China) at room temperature for 1 h. All specific bands were visualized using an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Optical densities were detected using ImageJ software version 1.48 (National Institute of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Apoptosis Markers
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Lysis of MC3T3-E1 cells took place in radio immunoprecipitation assay buffer with protease inhibitors (Beijing Solarbio Science & Technology Co., Ltd.), and centrifugation of lysates continued for 15 min at a temperature of 4°C at 9,600 × g so that precipitation can be removed. Subsequently, we assessed the amount of protein with the BCA assay kit (Thermo Fisher Scientific, Inc.). Equal amount of protein was added onto a 10 or 15% SDS-PAGE and transferred to a nitrocellulose membrane (EMD Millipore, Bredford, MA, USA). The membranes were blocked with 5% skim milk and probed with primary antibodies. After washing 3 times with phosphate-buffered saline (PBS), incubation of membranes with horseradish peroxidase conjugated secondary antibody (1:1,000; cat no. A0208; Beyotime Institute of Biotechnology, Shanghai, China) was conducted. We conducted western blotting tests via an enhanced chemiluminescence (ECL) kit (EMD Millipore). GAPDH was used as the internal standard. The sources of primary antibodies were as follows: Bax (Sc-493) and Bcl-2 (Sc-492) both from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); p53 (no. 2524) and GAPDH (no. 5174) both from Cell Signaling Technology, Inc. (Danvers, MA, USA).
6
Western Blot Analysis of Brain Protein
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The protein expressions in the brain were measured by the western blotting analysis. The brain tissue was lysed in radioimmunoprecipitation (RIPA) lysis buffer containing protease inhibitor cocktail at 4 °C for 30 min. The mixture was centrifuged at 12,000 rpm for 30 min, and the supernatants of mixture were slightly collected. The concentration of protein was determined Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) according to the Bradford method [54 (link)]. Equal amounts of proteins (15 μg) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto a polyvinylidene fluoride membrane. The membranes were incubated with 5% skim milk during 1 h, and were reacted with the primary antibodies such as Bax (sc-493, Santa Cruz Biotechnology Inc., Dallas, TX, USA), Bcl-2 (sc-492, Santa Cruz Biotechnology Inc.), and β-actin (#8457, Cell Signaling Technology Inc., Danvers, MA, USA) at 4 °C. After overnight, each membrane was incubated with secondary antibody at room temperature for 1 h. The protein bands were activated with enhanced chemiluminescence solution, and it was visualized with Davinch-chemiTM Chemiluminescence Imaging System (CoreBio, Seoul, Republic of Korea). The density of bands was quantified with Image J software (National Institutes of Health, Bethesda, MD, USA).
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