For immunofluorescence, 1/200 rabbit anti-YAP antibody (H-125, Santa Cruz), 1/400 rabbit anti-YAP/TAZ antibody (D24E4, Cell Signaling), 1/200 phalloidin-AlexaFluor 594 (Life Technologies), and 1/1000 anti-rabbit IgG-AlexaFluor 488 conjugate (Life Technologies) were used. For immunoblot, 1/3000 rabbit anti-YAP antibody (H-125, Santa Cruz), 1/3000 rabbit anti-TAZ antibody (#2149, Cell Signaling), 1/3000 rabbit anti-YAP/TAZ antibody (D24E4, Cell Signaling), 1/10,000 mouse anti-GAPDH antibody (6C5, Millipore), 1/5000 anti-mouse IgG-HRP (GE Healthcare), and 1/5000 anti-rabbit IgG-HRP (GE Healthcare), were used. Antibodies for Western blot were diluted in Can Get Signal reagents (Toyobo). Western blot using standard SDS–PAGE gel or gels containing Phos-tag-acrylamide (SuperSep Phos-tag, Wako) was performed as previously described [18] (link).
Can get signal reagent
Manufactured by Toyobo
Sourced in Japan
Can Get Signal reagents are a set of laboratory reagents designed for use in various analytical and experimental procedures. The core function of these reagents is to facilitate the detection and measurement of specific analytes or targets within a sample. The composition and application of these reagents are technical in nature, and a detailed description would require thorough knowledge of the underlying scientific principles and intended use cases.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg hrp, by GE Healthcare (2 mentions)
Anti rabbit igg hrp, by GE Healthcare (2 mentions)
Anti rabbit igg alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Phalloidin alexafluor 594, by Thermo Fisher Scientific (1 mentions)
H 125, by Santa Cruz Biotechnology (1 mentions)
Supersep phos tag, by Fujifilm (1 mentions)
Anti caspase 9 antibody, by Cell Signaling Technology (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Hydrochloric acid (hcl), by Fujifilm (1 mentions)
Diphenyl 1 pyrenylphosphine, by Dojindo Laboratories (1 mentions)
Acetic acid, by Fujifilm (1 mentions)
N acetyl l cysteine, by Fujifilm (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Immobilon western detection reagent, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Rabbit anti yap taz, by Cell Signaling Technology (1 mentions)
6 protocols using can get signal reagent
1
Immunofluorescence and Immunoblot Analysis of YAP/TAZ
2
Cell Viability and Oxidative Stress Assay Protocol
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The Cell Counting Kit-8 and Diphenyl-1-pyrenylphosphine (DPPP) were purchased from Dojindo (Tokyo, Japan), anti-monocarboxylate transporter 1 (MCT1) antibodies were obtained from Alpha Diagnostic International, Inc. (San Antonio, TX), and anti-caspase 9 antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA). Radioisotope of [2-14C]-Acetic acid sodium salt was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Acetic acid, hydrochloric acid and N-Acetyl-l -cysteine (NAC) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The 12% Bis Tris gels were obtained from Life Technologies Japan, Ltd. (Tokyo, Japan), and the Can Get Signal reagents were obtained from TOYOBO Co., Ltd. (Osaka, Japan). 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from LABOTEC Co., Ltd. (Tokyo, Japan).
3
Immunoblot Analysis of MAPK Signaling
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ST2L/EL-4 cells were lysed in RIPA buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS)) plus 20 mM β-glycerophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail (Roche). The protein samples were separated by electrophoresis on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were probed with antibodies against JNK, phosphorylated (p-) JNK, p38, p-p38, ERK, p-ERK (Cell Signaling Technology), and GAPDH (Santa Cruz Biotechnology), followed by horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology) and anti-mouse IgG antibodies (Bio-Rad Laboratories) in Can Get Signal reagents (Toyobo). The signals were detected with Immobilon western detection reagents (Millipore) and ImageQuant LAS4000 (GE Healthcare).
4
Western Blotting for Protein Analysis
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Western blotting was performed by standard methods. The following antibodies were used: 1/3000 rabbit anti‐YAP/TAZ (Cell Signaling Technology, Danvers, MA, USA; #8418), 1/10 000 mouse anti‐GAPDH (EMD Millipore, Burlington, MA, USA; MAB374), 1/3000 rabbit PAPR (Cell Signaling Technology; #9542), 1/5000 mouse anti‐E2F1 (Proteintech, Rosemont, IL, USA; 66515‐1‐Ig), 1/2000 rabbit anti‐FANCD2 (Proteintech; 204006‐1‐AP), 1/3000 mouse anti‐epidermal growth factor receptor (EGFR; Cell Signaling, #2239), 1/5000 anti‐mouse IgG‐HRP (GE Healthcare, Buckinghamshire, UK) and 1/5000 anti‐rabbit IgG‐HRP (GE Healthcare). All antibodies were diluted with Can Get Signal reagent (TOYOBO, Osaka, Japan). The band intensity was quantified by imagej (National Institute of Health, Bethesda, MD, USA), and the percentage of cleaved PARP was calculated as the percentages of cleaved PARP in total PARP: cleaved PARP/(full‐length PARP + cleaved PARP) × 100 in each sample.
5
Protein Analysis of EPA and NAC Effects
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EPA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 99% isopropanol (Wako, Tokyo, Japan) and stored at − 30 °C. For experiments, EPA was dissolved in serum-free Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 3% bovine serum albumin (BSA) (Sigma-Aldrich), and NAC (Wako) was dissolved in phosphate-buffered saline (PBS). Human phospho-kinase array (R&D Systems, Minnesota, MN, USA) and the human apoptosis antibody Array Membranes (Abcam, Cambridge, UK) were used for protein analysis. Antibodies against ERK1/2, phosphorylated (p)-ERK1/2, Pyk2, p-Pyk2, and β-actin were from Cell Signaling Technology Japan (Tokyo, Japan) and were diluted with Can Get Signal reagent (Toyobo, Osaka, Japan).
6
Apoptosis Assay of MLN8237 Treatment
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Cells were treated with 100 nM MLN8237 for 2 days. Cells were then washed with PBS and stained with Annexin V-APC (BioLegend, San Diego, CA, USA) and 10 μg/ml propidium iodide for 15 min. Cells were then analysed by FACSAria using FACSDiva software (BD, Franklin lake, NJ, USA).
Western blotting. Proteins from OVCAR-8 treated with 100 nM MLN8237 for 2 days were separated and blotted onto PVDF membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The blocking agent was 5% skimmed milk. Antibodies to the following were used: aurora-A (mouse, 1/10,000; Sigma), epidermal growth factor receptor (EGFR) (rabbit, 1/10,000; Proteintech, Rosemont, IL, USA), anti-YAP/TAZ (rabbit,1/3,000; Cell Signaling Technology, Danvers, MA, USA), phospho-histone H3 (rabbit; Cell Signaling Technology), mouse IgG-horseradish peroxidase (HRP) (1/5,000; GE Healthcare) and rabbit IgG-HRP (1/5,000; GE Healthcare). Antibodies were diluted with Can Get Signal reagent (Toyobo, Osaka, Japan).
Western blotting. Proteins from OVCAR-8 treated with 100 nM MLN8237 for 2 days were separated and blotted onto PVDF membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The blocking agent was 5% skimmed milk. Antibodies to the following were used: aurora-A (mouse, 1/10,000; Sigma), epidermal growth factor receptor (EGFR) (rabbit, 1/10,000; Proteintech, Rosemont, IL, USA), anti-YAP/TAZ (rabbit,1/3,000; Cell Signaling Technology, Danvers, MA, USA), phospho-histone H3 (rabbit; Cell Signaling Technology), mouse IgG-horseradish peroxidase (HRP) (1/5,000; GE Healthcare) and rabbit IgG-HRP (1/5,000; GE Healthcare). Antibodies were diluted with Can Get Signal reagent (Toyobo, Osaka, Japan).
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