AGS and NCI-N87 cells were washed with pre-cold PBS after 24 hours treatment with AT101 at 0.5, 2.5, and 5 μM, lysed with the RIPA buffer (50 mmol HEPES at pH 7.5, 150 mmol NaCl, 10% glycerol, 1.5 mmol MgCl2, 1% Triton-× 100, 1 mmol EDTA at pH 8.0, 10 mmol sodium pyrophosphate, 10 mmol sodium fluoride) containing the protease and phosphatase inhibitor cocktails, and centrifuged at 3000 × g for 10 minutes at 4°C. Protein concentrations were measured using Pierce BCA protein assay kit. Equal amount of protein sample (30 μg) was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer and electrophoresed on 7%–12% SDS-PAGE mini-gel after thermal denaturation at 100°C for 5 min. Proteins were transferred onto PVDF membrane at 400 mA for 1–2 hour at 4°C. Membranes were probed with indicated primary antibody overnight at 4°C and then blotted with respective secondary antibody. Visualization was performed using Bio-Rad ChemiDocTM XRS system (Hercules, CA, USA) with enhanced-chemiluminescence substrate and the blots were analyzed using Image Lab 3.0 (BioRad, Hercules, CA, USA). Protein level was normalized to the matching densitometry values of the internal control β-actin. The antibody against human β-actin was from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
Antibody against human β actin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Antibody against human β-actin is a laboratory reagent used for the detection and quantification of β-actin protein, a commonly used loading control in various cell and tissue analysis techniques. This antibody is specific to the human β-actin isoform and can be used in applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s phosphate buffered saline pbs, by Merck Group (3 mentions)
L arginine, by Merck Group (2 mentions)
L lysine, by Merck Group (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
13c6 l lysine, by Merck Group (2 mentions)
Annexin 5 phycoerythrin pe apoptosis detection kit, by BD (2 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (2 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Pierce bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Dmxaa, by Merck Group (1 mentions)
N4 l arginine, by Merck Group (1 mentions)
5 and 6 chloromethyl 2 7 dichlorodihydrofluorescein diacetate cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
Heat inactivated fetal bovine serum, by Merck Group (1 mentions)
Ionic detergent compatibility reagent, by Thermo Fisher Scientific (1 mentions)
Dialyzed fbs, by Merck Group (1 mentions)
Dmem medium, by Corning (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by Merck Group (1 mentions)
5 protocols using antibody against human β actin
1
Western Blot Analysis of AT101 Effects
2
Quantitative Proteomic Analysis of Apoptosis
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DMXAA (purity ≥ 98%), 13C6-L-lysine, L-lysine, 13C 15 (link)6 N4-L-arginine, L-arginine, RNase A, propidium iodide, Dulbecco’s phosphate-buffered saline (PBS), heat-inactivated fetal bovine serum (FBS), dialyzed FBS, and Roswell Park Memorial Institute (RPMI)-1640 medium for SILAC were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was sourced from Invitrogen Inc. (Carlsbad, CA, USA). A FASP™ protein digestion kit was purchased from Protein Discovery Inc. (Knoxville, TN, USA). RPMI-1640 medium for general cultural use was obtained from Corning Cellgro Inc. (Herndon, VA, USA). The polyvinylidene difluoride membrane was purchased from EMD Millipore Inc. (Bedford, MA, USA). Proteomic quantitation kits for acidification, desalting, and digestion, ionic detergent compatibility reagent, a Pierce bicinchoninic acid protein assay kit, and Western blotting substrate were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Primary antibodies against human cytochrome c, cleaved caspase 3, microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), LC3-II, and beclin 1 were all purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
3
Autophagy and Apoptosis Evaluation
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Cell lines were maintained in DMEM medium supplemented with 10% FCS were sourced from Corning Cellgro Inc (Herndon, VA, USA). A Cyto-ID autophagy detection kit was sourced from Enzo Life Sciences Inc (Farmingdale, NY, USA). 4, 6-Diamidino-2-phenylindole (DAPI) was obtained from Invitrogen (Carlsbad, CA, USA). The thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma-Aldrich Inc (St Louis, MO, USA). An Annexin V: phycoerythrin (PE) apoptosis detection kit was obtained from BD Biosciences Inc (San Jose, CA, USA). Primary antibodies against human were all purchased from Cell Signaling Technology Inc (Beverly, MA, USA). The antibody against human β-actin was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA).
4
Cisplatin-Induced Apoptosis and Autophagy
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CDDP was purchased from Selleckchem Inc. (Houston, TX, USA). 13C6-L-lysine, L-lysine, 13C615N4-L-arginine, L-arginine, Dulbecco’s modified Eagle’s medium (DMEM)/F12 for SILAC, APE1 siRNA, dimethyl sulfoxide (DMSO), 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, and Dulbecco’s phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 6-Diamidino-2-phenylindole (DAPI), Opti-minimal Essential Medium (MEM), Lipofectamine 2000, and the negative control siRNA were purchased from Invitrogen Inc. (Carlsbad, CA, USA). The Annexin V-phycoerythrin (PE) apoptosis detection kit was purchased from BD Biosciences Inc. (San Jose, CA, USA). The Cyto-ID® Autophagy detection kit was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The Western blotting substrate, Pierce™ bicinchoninic acid (BCA) protein assay kit, skim milk, and radioimmunoprecipitation assay buffer (RIPA) were purchased from Thermo Fisher Scientific Inc. (Hudson, NH, USA). The polyvinylidene difluoride (PVDF) membrane was obtained from Bio-Rad Inc. (Hercules, CA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The remaining primary antibodies for signalling proteins related to apoptosis and autophagy were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).
5
Investigating Nrf2, Autophagy, and Wnt Signaling
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Caff and Mel were purchased from Sigma-Aldrich Corp (St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Corning Cellgro Inc. (Manassas, VA, USA). G418 (geneticin, a selective antibiotic), Dulbecco’s phosphate-buffered saline (PBS), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich Corp. The autophagy detection kit was bought from Enzo Life Sciences Inc. (Farmingdale, NY, USA). Polyvinylidene difluoride (PVDF) membrane was purchased from EMD Millipore (Billerica, MA, USA). Western blot substrate was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies against human Nrf2, LC3-I/II, Beclin 1, Wnt3α, β-catenin, phosphorylated (p)-Tau/Tau, p-GSK3β/GSK3β, p-Akt/Akt, p-PI3K/PI3K (p85/55), and p-Erk1/2/Erk1/2 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
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