Cell lysates of melanoma cells (M14 and A375) were prepared using the RIPA Reagent (Cell Signaling Technology, Inc.). The protein concentration was quantified with bicinchoninic acid reagent (Sigma-Aldrich; Merck KGaA). A total of 30 µg protein samples were fractionated on 15% SDS-polyacrylamide gels and then transferred onto PVDF membranes. Thereafter, the membranes were blocked with 5% skimmed milk at room temperature for 2 h and probed with primary antibodies targeting SOX2 (1:1,000, cat. no. cat. no. ab171380; Abcam) or β-actin (1:1,000; cat. no. ab8227; Abcam) at 4˚C overnight. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:1,000; cat. no. ab205718, Abcam) at room temperature for 2 h. The bands were visualized using an ECL detection kit (Thermo Fisher Scientific, Inc.) and quantified using the ImageJ 1.8.0 software (National Institute of Health). The aforementioned antibodies used for this investigation were purchased from Abcam (Cambridge, MA, USA).
Ripa reagent
Manufactured by Cell Signaling Technology
Sourced in United States
RIPA reagent is a buffer solution used for protein extraction and cell lysis. It is designed to solubilize a wide range of proteins from various cell and tissue types. The reagent contains a combination of ionic and non-ionic detergents that help disrupt cell membranes and release intracellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti β catenin, by Cell Signaling Technology (2 mentions)
Depc solution, by Sangon (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Sybr premix ex taq 2 tli rnaseh plus, by Takara Bio (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Ab171380, by Abcam (1 mentions)
Bicinchoninic acid reagent, by Merck Group (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Steady glo luciferase assay system kit, by Promega (1 mentions)
Ellagic acid, by Chengdu Push Bio-technology (1 mentions)
Catechinic acid, by Chengdu Push Bio-technology (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
Gallic acid, by Chengdu Push Bio-technology (1 mentions)
Dapi fluoromount gtm, by Yeasen (1 mentions)
Alexa fluor 594 affinipure goat anti rabbit igg h l, by Yeasen (1 mentions)
Quantitative fluorescence imaging system, by LI COR (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Fluorescence conjugated secondary antibody, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using ripa reagent
1
Western Blot Analysis of SOX2 in Melanoma
2
Wnt3a Signaling Pathway Assay
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The powder of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from MP Biomedicals (Santa Ana, USA). DEPC solution was obtained from Sangon Biotech (Shanghai, China). Wnt3a protein was obtained from Stem RD (Burlingame, USA). Steady-Glo® luciferase assay system kit was from Promega Corporation (Madison, USA). PrimeSCript™RT reagent kit with gDNA Eraser and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) were purchased from Takara Bio Inc (Kusatsu, Shiga, Japan). The anti-β-catenin (#19807), anti-β-actin (#4970), anti-Bax (#14796), anti-Bcl-2 (#4223), anti-caspase3 (#9662), anti-PARP (#9532) and RIPA reagent were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The second antibody (#AP132P) was from EMD Millipore Corporation (Temecula, CA). Alexa Fluor 594 AFFINIpure Goat Anti-Rabbit IgG(H+L) and DAPI Fluoromount-Gtm were purchased from Yeasen Co., Ltd (Shanghai, China).
3
Phytochemical-Mediated Wnt Pathway Regulation
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Gallic acid (GA, 98%), ellagic acid (EA, 98%) and catechinic acid (CA, 98%) were from Chengdu PUSH Bio-technology Co., Ltd. (Chengdu, Sichuan, China). Ziyuglycoside I (98%) and ziyuglycoside II (98%) were purchased from Chengdu PureChem-Standard Co., Ltd (Chengdu, Sichuan, China). DEPC solution obtained from Sangon Biotech (Shanghai, China). Wnt3a protein was purchased from EMPOWERING STEM CELL R&D (Burlingame, CA, USA). The CCK-8 kit was obtained from Dojindo (Mashikimachi, Kamimashiki Gun Kumamoto, Japan) and Steady-Glo® Luciferase Assay System was from Promega Corporation (Madison, WI, USA). PrimeSCript™ RT reagent Kit with gDNA Eraser and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) were purchased from Takara Bio Inc (Kusatsu, Shiga, Japan). RIPA reagent, Anti-β-catenin (#19807) and anti-β-actin (#4970) were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Alexa Fluor 594 AFFINIpure Goat Anti-Rabbit IgG (H + L) and DAPI Fluoromount-Gtm were obtained from YEASEN Co., Ltd (Shanghai, China).
4
Western Blot Analysis of Phospho-ERK
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Proteins were extracted from HepG2 or PLC/PRF/5 cells using RIPA reagent (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease and phosphatase inhibitors (Thermo Fisher). Proteins were resolved by SDS-PAGE gradient gel (Thermo) and transferred to a PVDF membrane (Sigma). Membranes were incubated overnight with the following primary antibodies: anti-ERK (1:1000, CST), anti-p-ERK (1:1000, CST), and anti-tubulin (1:1000, CST). Following washing, membranes were incubated with fluorescence conjugated secondary antibodies (LI-COR). The immunoreactive bands were visualized by a quantitative fluorescence imaging system (LI-COR).
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