Radioimmunoprecipitation Assay buffer (Sigma-Aldrich; Merck KGaA) as well as Protease Inhibitor (Sigma-Aldrich; Merck KGaA) were used to lyse cells (28 (link)). Following protein collection, a BCA Pierce Assay (Thermo Fisher Scientific, Inc.) was used to quantify the protein concentration. Protein (50 µg) from each sample was denatured and resolved by 10% SDS-PAGE (EMD Millipore, Billerica, MA, USA) and electroblotted to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore). PVDF membranes were incubated with 5% non-fat milk for one hour at room temperature and were then incubated with anti-TSPAN1 antibody (1:300; cat.no. NBP2-33867; Novus Biologicals, LLC, Littleton, CO, USA) or anti-β-actin antibody (1:1,000; cat.no. NB600-503; Novus Biologicals, LLC) at 4°C overnight. Following washing with TBS with 5% Tween-20, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000; cat.no. NB710-57836; Novus Biologicals, LLC) for 1.5 h at room temperature. Finally, signals were developed by enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.). The optical density of each protein band was quantified by a scanning densitometer and Quantity One software (version 4.4.1; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each lane of protein band density was normalized against corresponding β-actin densities.
Nb600 503
Manufactured by Novus Biologicals
Sourced in United States
The NB600-503 is a high-quality lab equipment product offered by Novus Biologicals. It is designed to perform specific laboratory functions. The core function of this product is to facilitate precise and reliable measurements or analysis in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Nbp2 33006, by Novus Biologicals (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Azure 300 chemiluminescent imaging system, by Azure Biosystems (1 mentions)
Ab34173, by Abcam (1 mentions)
West pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
Optizen nanoq, by Mecasys (1 mentions)
Anti runx2, by Santa Cruz Biotechnology (1 mentions)
Scanjet 4c, by Hewlett-Packard (1 mentions)
6 protocols using nb600 503
1
Western Blot Analysis of TSPAN1 Protein
2
Western Blotting of Pluripotent Stem Cells
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Protein samples for western blotting were isolated from culture-adapted PSCs by homogenization with lysis buffer (#9803, CST, USA). The samples were boiled in laemmli buffer (#161-0737, Bio-Rad, USA) for 5 min and were resolved on a 4%-12% gradient SDS-PAGE gel and blotted to PVDF membrane. Following overnight incubation with primary antibodies against α-SMA (1:1000; #NBP2-33006, Novus Biologicals, USA), GFAP (1:1000; #60190-1-Ig, Proteintech, USA) and beta-actin (1:1000, # NB600-503, Novus Biologicals, USA) and detection by HRP-conjugated IgG. Bands were visualized using an Azure 300 chemiluminescent imaging system (Azure Biosystems, USA).
3
Western Blot Analysis of NQO-1
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Briefly, distal colon tissue sections were homogenised and lysed in a RIPA buffer containing protease inhibitor cocktails (Complete ULTRA Tablets, Mini, EDTA-free, Roche, New South wales, Australia). Protein quantification was done using a DC protein Assay Kit from Biorad. The samples were suspended in loading dye and boiled at 95 °C for 5 min. A total of 20 μg of protein was separated on 4–15% of SDS-PAGE gel (Mini-PROTEAN TGX Precast Gels (50 µL), Biorad, New South Wales, Australia) at 100 V for 60 min, then electro-transferred to a PVDF membrane at 250 A for 60 min. The membranes were blocked with 5% non-fat milk for 1 h at room temperature (RT) and then incubated with primary antibodies against NQO-1 (1:1000) (ab34173, Abcam) and β-actin (1:8000) (NB600-503, Novus Biologicals) overnight at 4 °C. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000, 7074, Cell Signaling Technology, Australia) for 1 h at RT. The bands were visualised with a chemiluminescence reagent (SuperSignal, West Pico PLUS, Chemiluminescent Substrate, Thermo Scientific, Victoria, Australia) and imaged using a Fujifilm Luminescent Image Analyzer (LAS-3000 image reader, version 2.2) (Fuji Life Sciences, Japan).
4
Protein Isolation from Tissue Samples
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For protein isolation, tissue powders in each replicate were homogenized by a Daihan 15D tissue homogenizer (27,000 rpm) in 400 μL of RIPA Lysis Buffer (including 2 μL of PMSF, 2 μL of a sodium orthovanadate, and 2 μL of a protease inhibitor cocktail) (RIPA Lysis Buffer System, sc-24948; Santa Cruz, CA, USA) under ice-cold conditions. The lysate was centrifuged at 14,000 g for 20 minutes and in the range of 2.13–3.17 mg/mL of total protein was detected by the protein A280 method with a micro-volume spectrophotometer (Optizen Nano Q; Mecasys). The protein level was measured using a Western blot assay and SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) electrophoresis, as described by Delen et al. [15 (link)] with two primary antibodies (DDR1 [NBP1-33134; Novus Biologicals, Littleton, CO, USA] and DDR2 [NBP2-14927; Novus Biologicals]) and normalized with a beta-actin antibody (NB600-503; Novus Biologicals). Protein quantities were determined with the ProteinQuant software and given as relative density.
5
Western Blot Analysis of EXO1 Protein
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Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer plus protease inhibitors. Lysates were incubated in RIPA for 15 min at 4°C and centrifuged at 4°C for 15 min. Protein concentrations were assessed using a Bradford protein assay. Samples were then resuspended in 4× lithium dodecyl sulfate sample buffer and denatured using heat. Protein samples were electrophoresed on 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes in transfer buffer (20% methanol, 11% glycine and 3.4% Tris) at 100 V. Membranes were blocked with 5% milk in Tris-buffered saline–0.1% Tween (TBST) at room temperature for 45 min. The membranes were then incubated overnight in 5% milk in TBST at 4°C with an EXO1 (Thermo Fisher, 266) or actin (Novus, NB600-503) antibody. Membranes were subsequently washed in TBST and exposed to horseradish peroxidase-conjugated secondary antibodies in 5% milk in TBST at room temperature for 1–2 h. Membranes were subsequently developed and enhanced using a commercial enhanced chemiluminescent kit.
6
Western Blot Analysis of Runx2, α-actin, and β-actin
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Cell monolayers were lysed with Laemmli's buffer (Laemmli, 1970) . Total protein content of the cell lysates was evaluated by a micro-method (Lowry et al., 1951) . Lysates were heated to 100 C for 3 min, after which aliquots containing 40 mg of protein were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were then transferred to PVDF membranes (Millipore, Bedford, MD), which were blocked in 3% non-fat dry milk in Tris-buffered saline (TBS) for 2 h at room temperature. They were then incubated at 4 C for 24 h with anti-Runx2(Santa Cruz Biotechnologies, sc-10758, Lot #H1909), anti-aactin (Santa Cruz Biotechnologies, sc-53142, Lot #H1909), or anti-bactin polyclonal antibodies (Novus Biologicals, NB600-503, Lot # A5) diluted 1:2000 in PBS with 0.5% bovine serum albumin. After four washes in PBS with 0.1% Tween 20, the membranes were incubated with a secondary goat anti-rabbit antibody, followed by staining with the peroxidase-biotin reagent and diaminobenzidine from the Vectastain kit. The intensity of the Runx2, a-actin and bactin specific bands was quantified by densitometry after scanning the PVDF membrane with a Hewlett-Packard Scanjet 4C. Images were analysed using the gel plugin of Image J program (www. macbiophotonics.ca/imagej).
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