L-α-Phosphatidylcholine from soybean (SPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti polar lipids (Alabaster, AL, USA). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-Mal) was obtained from LaySan Bio (Arab, AL, USA). Erbitux 2 mg/mL (Cetuximab) was a kind gift from cancer hospital clinical pharmacy. 5-Fluorouracil (5-FU), Traut’s reagent and 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Precision Plus Protein Dual Color Standards, Ready gel Tris-HCL precast gels, Laemmli sample buffer 4X, 2-mercaptoethanol and Tris/ Glycine/ SDS buffer 10X were purchased from Biorad (Hercules, CA, USA) and used according to manufacturer’s instructions. Sepharose CL-4B and disposable PD-10 desalting columns were obtained from GE Healthcare (Pittsburgh, PA, USA). Antibiotic/antimycotic solutions, fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Grand Island, NY). Bicinchoninic acid protein assay (BCA) kit was obtained from Thermo Scientific (Pittsburg, PA, USA). A431 cells (ATCC® CRL1555™) were purchased from American Type Culture Collection (ATCC) and cultivated according to the instructions.
Ready gel tris hcl precast gels
Manufactured by Bio-Rad
Sourced in United States
Ready Gel Tris-HCl Precast Gels are laboratory equipment designed for protein electrophoresis. They provide a pre-cast gel matrix for the separation and analysis of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop phosphatase inhibitor cocktail tablet, by Roche (2 mentions)
Immobilon p filter, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Sepharose cl 4b, by GE Healthcare (1 mentions)
Laemmli sample buffer 4x, by Bio-Rad (1 mentions)
Disposable pd 10 desalting column, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Bio-Rad (1 mentions)
1 2 distearoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
L α phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
1 4 5 dimethylthiazol 2 yl 3 5 diphenylformazan mtt, by Merck Group (1 mentions)
A431 cells atcc crl1555, by American Type Culture Collection (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Imagequant las 4000, by Fujifilm (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
3 protocols using ready gel tris hcl precast gels
1
Preparation and Characterization of Cetuximab-Conjugated Liposomes
2
Western Blot Analysis of ABCC10/MRP7
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Cells were lysed in sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 1 mM EDTA, and 10% glycerol) with Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Mannheim, Germany) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche Diagnostics). Subsequently, equal amounts of protein were applied to 7.5% Ready Gel Tris-HCl Precast Gels (Bio-Rad Laboratories, Hercules, CA) and electrophoresed. Then, that transferred onto Immobilon-P filters (Millipore, Billerica, MA). The filters were first incubated with primary antibodies against ABCC10/MRP7 (160 kDa) and α-tubulin (50 kDa) overnight at room temperature and then with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Chemiluminescence images were captured on ImageQuant LAS4000 (Fujifilm, Tokyo, Japan). The following antibodies were used: anti-ABCC10/MRP7 (MyBiosource, San Diego, CA), anti-α-tubulin (Sigma Aldrich Biotechnology, St. Louis, MO) and HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA). α-tubulin was used as a loading control. The band intensities were analyzed by Image Quant TL (GE Healthcare Bioscience, Amersham Place, UK).
3
Western Blot Analysis of Nestin and Tubulin
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Equal amounts of total cell lysates were solubilized in sample buffer (50 mM Tris-HCL (pH 6.8), 2% SDS, 1 mM EDTA, and 10% glycerol) with Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Mannheim, Germany) and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche Diagnostics). Subsequently, these lysates were electrophoresed on 4–20% Ready Gel Tris-HCl Precast Gels (Bio-Rad Laboratories, Hercules, CA), then transferred onto Immobilon-P filters (Millipore, Billerica, MA). These filters were first incubated with primary antibodies against nestin (155 kDa) and α-tubulin (50 kDa) at room temperature overnight, then with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The following antibodies were used: anti-nestin (clone 10C2) (Millipore), anti-α-tubulin (Sigma Aldrich Biotechnology, St. Louis, MO) and HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA). The specific antibody-antigen complexes were detected using an ECL detection system (GE Healthcare Bioscience, Fairfield, CT). This study used α-tubulin as a loading control.
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