Aristolochic acid (AA), 3-methylcholanthrene (MCA), 3-(4,5-dimethylthiazol- 2-yl)-2,5- diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Coomassie Blue R-250, Ponceau S,ERK1/2 inhibitor (PD98059), p38MAPK inhibitor (SB203580), NaCl, NaN3, ZnCl2, CaCl2, Triton-X 100, and rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase-conjugated IgG and PVDF (polyvinylidenefluoride) membranes were obtained from Millipore (Bellerica, MA, USA). A chemiluminescent HRP substrate was purchased from Pierce (Rockford, IL, USA). Rabbit anti-human mitogen-activated protein kinase kinase3 (MKK3), MAPK kinase kinasekinase4 (MEKK4), focal adhesion kinase (FAK), and growth factor receptor-bound protein 2 (GRB2) antibodies were obtained from Epitomics (Burlingame, CA, USA). Rabbit anti-human TIMP-1 and TIMP-2 antibodies were obtained from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), uPA, c-jun N-terminal kinase (JNK), phosphorylated c-jun N-terminal kinase (p-JNK), extracellular signal regulated kinases (ERK), phosphorylated extracellular signal regulated kinases (p-ERK), p38 and p-p38 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).
Rabbit anti human β actin antibody
Manufactured by Merck Group
Sourced in United States
Rabbit anti-human β-actin antibodies are a laboratory product used for the detection and quantification of the β-actin protein in human samples. β-actin is a commonly used housekeeping gene and its protein is involved in the structure and function of the cytoskeleton. These antibodies can be used in various immunoassay techniques to measure the expression levels of β-actin in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Z devd fmk, by Merck Group (4 mentions)
P erk, by Cell Signaling Technology (4 mentions)
P mtor, by Cell Signaling Technology (3 mentions)
Goat anti rabbit and horseradish peroxidase conjugated igg, by Merck Group (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Cleaved caspase 9, by Cell Signaling Technology (3 mentions)
Cytochrome c, by Cell Signaling Technology (3 mentions)
P perk, by Cell Signaling Technology (3 mentions)
P pi3k, by Cell Signaling Technology (3 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (3 mentions)
Z vad fmk, by Merck Group (2 mentions)
Procaspase 9, by Cell Signaling Technology (2 mentions)
P jnk, by Proteintech (2 mentions)
Procaspase 3, by Cell Signaling Technology (2 mentions)
Bcl xl, by Cell Signaling Technology (2 mentions)
Mcl 1, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
13 protocols using rabbit anti human β actin antibody
1
Signaling Pathways Modulation in Cells
2
Purification of 13-Acetoxysarcocrassolide from Soft Coral
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13-Acetoxysarcocrassolide was purified after extracting it from the cultured Formosa soft coral S. crassocaule, according to the method described by others (Duh et al. 2000 (link)). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Z-DEVD-FMK (caspase-3 inhibitor), Z-VAD-FMK (a cell-permeable pan-caspase inhibitor), a protease inhibitor cocktail, and rabbit anti-human β-actin antibodies were purchased from Sigma (St. Louis, MO, USA). Antibodies against pro-caspase 9, cleaved-caspase 9, pro-caspase 3, cleaved-caspase 3, cleaved-PARP-1, Bax, Bad, Bcl-xL, Mcl-1, p-Bad, PARP-1, Bcl-2, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, p70S6K, p-p70S6K, 4EBP1, p-4EBP1, p-S6, p-eIF4B, and p-eIF4E, and goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin G were obtained from Cell Signalling Technology (Danvers, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were obtained from Biowest (Nuaillé, France).
3
Western Blot Analysis of Apoptosis Markers
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Rabbit anti-human Bax, ERK, JNK and p-JNK antibodies were obtained from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against p-PI3K, AKT, p-AKT, PARP, pro-caspase-3, cleaved-caspase-3, pro-caspase-9, cleaved-caspase-9, Bcl-xl, Bcl-2, Mcl-1, Bad, p38, p-p38, p-ERK, p-ATF2 and p53 were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human antibodies against p-Bad and cytochrome C were obtained from Epitomics (Burlingame, CA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG was obtained from Millipore (Bellerica, MA, USA). Immunoblotting was performed as previously described [19 (link)]. The treated samples and the control samples (25 μg) were separated by 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins on the gel were then transferred to PVDF membranes. The PVDF membranes were incubated with the primary antibody (1:1000 dilutions in 2% dehydrated skim milk) at 4 °C overnight. Then, incubation at 4 °C was performed for 2 h with the secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk). The blots were detected through chemiluminescence using enhanced ECL western blotting kit.
4
Molecular Mechanisms of Apoptosis Regulation
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Nobiletin, Z-VAD-FMK, Z-DEVD-FMK, dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) protease inhibitor cocktail, salubrinal, rabbit anti-human β-actin antibodies, and Dulbecco’s modified Eagle′s medium (DMEM) were purchased from Sigma (St. Louis, MO, USA). An annexin V-FITC/PI apoptosis detection kit was purchased from Pharmingen (San Diego, CA, USA). Antibodies against pro-caspase 9, cleaved caspase 9, caspase 8, pro-caspase3, cleaved caspase 3, Mcl-1, Bax, Bcl-xl, Bad, p-Bad, PARP-1, Bcl-2, 14-3-3, PDI, GRP78, calreticulin, cytochrome C, p-PERK, PERK, p-eIF2α, eIF2 α, ATF6-f, ATF4, IRE-1α, CHOP, p-JNK, p-c-jun, JNK, p-JNK, MAPKp38, p-MAPKp38, ERK, p-ERK, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, and goat anti-rabbit and horseradish peroxidase-conjugated immunoglobulin (Ig) G were obtained from Cell Signaling Technology (Danvers, MA, USA). Fetal bovine serum (FBS) was obtained from Biowest (Nuaillé, France).
5
Protein Expression Analysis of Flaccidoxide-13-acetate
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Rabbit anti-human ERK, p-ERK, JNK, p-JNK, GRP78, ATF4, and cleaved-ATF6 antibodies were purchased from ProteinTech Group (Chicago, IL, USA). Rabbit anti-human antibodies against AKT, p-AKT, PI3K, p-PI3K, Mcl-1, Bad, p-Bad, Bcl-xl, Bcl-2, Bax, p38, p-p38, PERK, p-PERK, elF2α, p-elF2α, pro-caspase 3, cleaved caspase 3, pro-caspase 9, cleaved caspase 9, cytochrome C, and CHOP were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Cytosolic cytochrome C were separated using a cytochrome C releasing apoptosis assay kit (Biovision, Milpitas, CA, USA).
The flaccidoxide-13-acetate treated sample and DMSO treated control samples (total protein 25 μg) were separated by 12.5% SDS-PAGE, and the proteins on the gel were transferred to a PVDF membrane. The membrane containing transferred protein was blocked in PBS buffer and incubated with primary antibody at 4 °C overnight, followed by secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk) for 2 h at 4 °C. The signals were detected with an enhanced chemiluminescence detection kit.
The flaccidoxide-13-acetate treated sample and DMSO treated control samples (total protein 25 μg) were separated by 12.5% SDS-PAGE, and the proteins on the gel were transferred to a PVDF membrane. The membrane containing transferred protein was blocked in PBS buffer and incubated with primary antibody at 4 °C overnight, followed by secondary antibodies (goat anti-rabbit or goat anti-mouse and horseradish peroxidase conjugate, 1:5000 dilution in 2% dehydrated skim milk) for 2 h at 4 °C. The signals were detected with an enhanced chemiluminescence detection kit.
6
Piper betle Stem Bioactivity Analysis
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The stems of Piper betle were collected in Pingtung County, Taiwan in July 2008, which were cultivated by local farmer and bornyl cis-4-hydroxycinnamate identified by Chi-I Chang, National Pingtung University of Science and Technology. Rabbit anti-human MMP-2, MMP-9, uPA, TIMP-1, TIMP-2, FAK, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, JNK, p-JNK, Jun, p-Jun, p38, p-p38, ERK, and p-ERK were obtained from Cell Signaling Technology (Danvers, MA, USA). GRB2, Rac, PKC, Ras, RhoA, MEKK3, MEKK7, N-cadherin, E-cadherin, Snail, and Lamin A2 antibodies were obtained from Epitomics (Burlingame, CA, USA). Dimethyl sulfoxide (DMSO), protease inhibitor cocktail, and rabbit anti-human β-actin antibodies were purchased from Sigma (St. Louis, MO, USA). PVDF (polyvinylidene difluoride) membranes and goat anti-rabbit and horseradish peroxidase-conjugated IgG were purchased from Millipore (Bellerica, MA, USA). Chemiluminescent horseradish peroxidase (HRP) substrate was purchased from Pierce (Rockford, IL, USA).
7
Extraction and Analysis of 13-AC from Soft Coral
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13-AC was extracted from cultured Formosa soft coral Sarcophytoncrassocaule following the protocol described elsewhere [63 (link)] and dissolved in DMSO. A mitochondria/cytosol fractionation kit was obtained from BioSource International (Camarillo, CA, USA). Chemiluminescent HRP substrate was purchased from Pierce (Rockford, IL, USA). An annexin V–FITC Apoptosis Detection kit was obtained from Pharmingen (San Diego, CA, USA). A JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide) fluorescent kit was obtained from Biotium (Hayward, CA, USA). A DeadEnd™ Fluorometric TUNEL fluorescent kit and a 4′-6-diamidino-2-phenylindole (DAPI) fluorescent kit were obtained from Promega (Madison, WI, USA). 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), cyclosporine A (CyA), aristolochic acid (ArA), trifluoperazine (TFZ), Z-DEVD-FMK (caspase-3 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), protease inhibitor cocktail and rabbit anti-human β-actin antibodies were obtained from Sigma (St Louis, MO, USA). Goat anti-rabbit and horseradish peroxidase conjugated IgG and PVDF (polyvinylidenedifluoride) membranes were obtained from Millipore (Bellerica, MA, USA).
8
Apoptosis and Autophagy Regulation
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Reagents Dulbecco's modified Eagle's medium (DMEM), trypsin-ethylenediaminetetraacetic acid, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were obtained from Biowest (Nuaillé, France). Polyvinylidene difluoride (PVDF) membranes, goat anti-rabbit, and horseradish peroxidase-conjugated immunoglobulin (Ig) G were obtained from Millipore (Billerica, MA, USA). Protease inhibitor cocktail, DMSO, salubrinal (Sal), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ROS Green Flow Cytometry Assay Kit, Z-DEVD-FMK (caspase-3 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), 3-methyladenine (3-MA), and rabbit anti-human β-actin antibodies were obtained from Sigma (St. Louis, MO, USA). Cell extraction RIPA buffer was obtained from TOOLS (Taiwan). Enhanced chemiluminescence (ECL) Western blotting reagents were obtained from Pierce Biotechnology (Rockford, IL, USA). Antibodies against Beclin-1, LC3-I, LC3-II, Atg3, Atg5, Atg7, Atg12, and Atg16 were obtained from Epitomics (Burlingame, CA, USA). Antibodies against procaspase 3, cleaved-caspase 3, procaspase 9, cleaved-caspase 9, cytochrome c, Bax, Bad, p-Bad, Bcl-2, Bcl-xl, Mcl-1, GRP78, CALR, elF2α, p-elF2α, IRE1α, PERK, p-PERK, ATF4, ATF6-f, CHOP, and PARP1 were obtained from Cell Signaling Technology (Danvers, MA, USA).
9
Investigating the mTOR Signaling Pathway
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PBS buffer, Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum, as well as anti-mTOR, anti-Akt, anti-p70S6K, anti-p-mTOR (Ser 2448), anti-p-Akt (Ser473), and anti-p-p70S6K (Thr389) antibodies were obtained from Cell Signaling Technology (MA, USA). Dimethyl sulfoxide (DMSO), protease inhibitor cocktail, and rabbit anti-human β-actin antibodies were obtained from Sigma (St. Louis, MO, USA). PCR kit, Trizol, and first-strand cDNA synthesis kit were purchased from TAKARA Company (Dalian, China). Polyvinylidene difluoride (PVDF) membranes and goat anti-rabbit and horseradish peroxidase-conjugated IgG were obtained from Millipore (MA, USA). TRIzol reagent and sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gels were acquired from Beyotime Biotechnology (Haimen, China).
10
Proteomic Identification of Protein Markers
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Protein spots on the 2-DE gel were visualized after CBR staining. Spots of interest were then excised into a piece (1 mm × 1 mm) from the 2-DE gel and digested by trypsin. The in-gel digested sample was identi ed by LC-MS/MS using an AB SCIEX QTRAP 5500Q mass spectrometer (Applied Biosystems, CA, USA). The detailed procedure was described in a previous study (17) .
Western blot analysis 1-DE or 2-DE PAGE samples were transferred to a PVDF membrane (Millipore) under 0.4 A for 2 h using Transphor TE 62 (GE Healthcare, Chicago, IL, USA). The membrane was rst incubated with rabbit anti-human CRT and AnxA2 (ProteinTech Group, Chicago, IL, USA) or rabbit anti-human β-actin antibodies (Sigma, St. Louis, MO, USA), then with goat anti-rabbit or rabbit anti-mouse horseradish peroxidase-conjugated IgG (1/5,000 dilution, Millipore, Bellerica, MA, USA) at room temperature for 2 h. After washing with PBST three times, the protein concentrations were determined from the chemiluminesence intensity using Pierce™ ECL Western blotting reagents (Thermo Fisher Scienti c, Waltham, MA, USA).
Western blot analysis 1-DE or 2-DE PAGE samples were transferred to a PVDF membrane (Millipore) under 0.4 A for 2 h using Transphor TE 62 (GE Healthcare, Chicago, IL, USA). The membrane was rst incubated with rabbit anti-human CRT and AnxA2 (ProteinTech Group, Chicago, IL, USA) or rabbit anti-human β-actin antibodies (Sigma, St. Louis, MO, USA), then with goat anti-rabbit or rabbit anti-mouse horseradish peroxidase-conjugated IgG (1/5,000 dilution, Millipore, Bellerica, MA, USA) at room temperature for 2 h. After washing with PBST three times, the protein concentrations were determined from the chemiluminesence intensity using Pierce™ ECL Western blotting reagents (Thermo Fisher Scienti c, Waltham, MA, USA).
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