Protein extracts from DU145 and PC-3 cells were prepared in Mammalian Cell Lysis/Extraction Reagent (Sigma-Aldrich). The protein concentration was determined using the Bradford reagent (Bio-Rad). Equal amounts of protein lysate were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% non-fat milk powder in PBS-Tris buffer for 1 h, followed by incubation with primary antibody at 4°C overnight. Membranes were then incubated with the corresponding secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. Enhanced chemiluminescence Western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA) were used for protein detection. The X-ray films were scanned and bands were analyzed.
Mammalian cell lysis extraction reagent
Manufactured by Merck Group
The Mammalian Cell Lysis/Extraction Reagent is a solution designed to facilitate the lysis and extraction of cellular contents from mammalian cells. It is a buffer formulation that aids in the disruption of cell membranes and the release of intracellular components, including proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence western blotting detection reagent, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by Beyotime (1 mentions)
Bca kit for protein determination, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Ecl western blotting kit, by Beyotime (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Mini protein tetra system, by Bio-Rad (1 mentions)
5 protocols using mammalian cell lysis extraction reagent
1
Western Blot Analysis of Prostate Cancer Cells
2
Quantitative Western Blot Analysis
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After specific treatment, total proteins were isolated with a mammalian cell lysis/extraction reagent (Sigma-Aldrich Co.) according to the manufacturer’s protocol. An equal amount of proteins were separated on the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to polyvinylidene fluoride membranes. After blocking, the membranes were incubated with specific primary antibodies at 4°C overnight. Then, the membranes were washed with tris-buffered saline with Tween-20 (TBST) for three times for 15 min each. After incubating with secondary antibodies for 45 min at 37°C and washing with TBST, an ECL kit (EMD Millipore, Billerica, MA, USA) was used to visualize membrane immunoreactivity. Quantification was performed using a computerized imaging program Quantity One (Bio-Rad Laboratories Inc., Hercules, CA, USA).
3
Protein Expression Analysis in Cell Lines
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Protein lysate from CAFs, NFs, SKOV3 and HO-8910 cells were prepared in Mammalian Cell Lysis/Extraction Reagent (Sigma, St. Louis, MO) supplemented with 1% Triton X-100 and 1% protease inhibitor Cocktail. Total protein concentration was determined by BCA Kit for Protein Determination (Sigma–Aldrich, Cat# BCA1). Equal amount of protein lysate were resolved by SDS/PAGE and transferred on to the PVDF membrane (Millipore, Billerica, MA) for Western blotting analysis with the corresponding primary and secondary antibodies (Table 1 ). ECL Western blotting detection reagents (Beyotime, Cat#P0018-1/-2) were used for protein detection. The X-ray films were scanned and bands were analysed.
4
Caco-2 Cell Protein Extraction and Immunoblotting
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Caco-2 cells were homogenized in Mammalian Cell Lysis/Extraction Reagent (Sigma, St. Louis, MO) supplemented with 1% protease inhibitor cocktail and 1% Triton X-100 (TX-100), and lysates were obtained. Protein concentrations were measured using a BCA Protein Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein lysate (35 micrograms) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes for WB analysis with the primary antibodies and corresponding secondary antibodies (Supplementary Table 3 ). An ECL Western blotting kit from Beyotime Biotechnology was used for the detection of proteins. Subsequently, the bands on X-ray films were scanned and analysed.
5
Western Blot Analysis of Signaling Pathways
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Total cell lysates were prepared with Mammalian Cell Lysis/ Extraction Reagent (Sigma; 1% Triton X-100 and 1% protease inhibitor Cocktail were added) followed by incubating the plate on ice for 30 min. The lysates were collected and centrifuged at 12,000g for 10 min at 4°C, and the supernatant was recovered and placed into a fresh tube. Samples were boiled and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% acrylamide), and then proteins were blotted onto nitrocellulose membranes (Millipore) using electrotransfer system (mini-protein Tetra System; BioRad) in a buffer solution containing 193 mM glycine, 25 mM Tris (pH 8.3), and 20% methanol. The blots were then probed with P-tyrosine, FGFR4, ERK1/2, phospho-ERK1/2, p38, phosphor-p38, c-jun, phospho-c-jun, Ecad, Snail, MMP3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibodies. Bound primary antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies and the bound conjugate was then detected using the enhanced chemiluminescence (ECL) detection system. Equal protein loading was assessed by the expression of GAPDH.
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