ASMCs were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The extracts were collected and protein concentrations were measured using a bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology, Ltd.). Equal quantities (40 μg) of protein were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Wuhan Boster Biological Technology, Ltd.) and blotted onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Bedford, MA, USA). The PVDF membrane was blocked for nonspecific protein binding with Tris-buffered saline/Tween-20 (TBST; Guangzhou Whiga Technology Co., Ltd.) and 5% non-fat milk (Guangzhou Whiga Technology Co., Ltd.) at room temperature for 2 h. Then, the membrane was incubated overnight at 4°C with primary goat anti-TRPM7 (1:500; Abcam, Cambridge, UK) and primary mouse anti-β-actin (1:1,000; Sigma-Aldrich, St. Louis, MO, USA) monoclonal antibodies. Next, the membranes were washed with TBST, incubated with HRP-conjugated rabbit anti-goat IgG (1:500) and rabbit anti-mouse IgG (1:1,000) secondary antibodies (Cell Signaling Technology, Inc.) for 1 h, and then washed three times with TBST. The proteins were detected using an enhanced chemiluminescence system (a SignalBoost™ Immunoreaction Enhancer kit; Merck Millipore).
Bicinchoninic acid protein assay kit
Manufactured by Wuhan Boster Biological Technology
Sourced in China
The Bicinchoninic acid (BCA) protein assay kit is a colorimetric detection and quantitation assay for protein. It utilizes the well-known reduction of Cu2+ to Cu+ by protein in an alkaline medium, with the Cu+ ions detected using a reagent containing bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Abcam (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Radioimmunoprecipitation assay protein lysis buffer, by Beyotime (2 mentions)
Ecl assay kit, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Rabbit anti mouse igg, by Cell Signaling Technology (1 mentions)
Signalboost immunoreaction enhancer kit, by Merck Group (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
P akt, by Abcam (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
P pi3k, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Media Cybernetics (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
9 protocols using bicinchoninic acid protein assay kit
1
Western Blot Analysis of TRPM7 Protein Levels
2
Protein Expression Analysis in Aortic Tissues
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The total protein of aortic tissues in each group was extracted. The protein concentration was determined according to the bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). The extracted protein added with uploading buffer and separated with 10% polyacrylamide gel electrophoresis (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). The proteins were transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h. Primary antibodies of Caspase-3, Bax, Bcl-2, VEGF, PI3K, p-PI3K, AKT, p-AKT and β-actin (1: 3000, Abcam, Cambridge, MA) were added and incubated at 4 °C overnight, followed by washing three times (5 min per wash) with Tris-buffered saline with Tween 20 (TBST). Corresponding secondary antibodies (Shanghai Miaotong Biotechnology Co., Ltd., Shanghai, China) were added and incubated for 1 h. The membranes were washed for three times with 5 min for each time. Chemiluminescence reagents were employed to develop images. β-actin was considered as an internal reference. The images of the gels were captured in a Bio-Rad Gel Doc EZ Imager (Bio-Rad, Hercules, CA). The gray values of target protein bands were analyzed by ImageJ software (National Institutes of Health, Bethesda, MA). The experiment was conducted in triplicate.
3
Western Blot Analysis of Apoptotic Markers
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Following the treatments, total protein from the cells was extracted using radio-immunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) containing 60 µg/ml phenylmethylsulfonyl fluoride. Protein concentrations were assessed using a bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China). Proteins were separated on SDS-polyacrylamide gel, then transferred to a nitrocellulose membrane and incubated overnight at 4°C with the following antibodies: Rabbit monoclonal anti-procaspase 3 (1:1,000; cat. no. 9665), anti-cleaved caspase 3 (1:1,000; cat. no. 9664), anti-poly ADP ribose polymerase (PARP; 1:1,000; cat. no. 9532) and anti-cleaved PARP (1:1,000; cat. no. 5625) and mouse monoclonal anti-β-actin (1:5,000; cat. no. 3700) (Cell Signaling Technology, Inc., Beverly, MA, USA). Blots were washed four times with TBS containing 0.1% Tween-20 (TBST), and then incubated with horseradish peroxidase (HRP)-linked horse anti-mouse and anti-rabbit secondary antibodies (1:200; cat. no. 7076 and 7074S respectively; Cell Signaling Technology, Inc.) at room temperature for 1 h. Antibody binding was detected using a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology, Inc., Rockford, IL, USA) according to the manufacturer's instructions.
4
ALP Activity Assay for Cell Differentiation
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Cell differentiation on the titanium disks after 7 days was detected using an ALP activity assay. The cells were lysed with 1% Triton-X 100. Then, protein concentrations were measured with a bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China). The absorbance values were measured at 520 nm with a microplate reader, and ALP activity was calculated according to the absorbance values.
5
Western Blot Analysis of NF-κBp65 in NP Tissues
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Protein samples were prepared from NP tissues using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein concentration was measured using a bicinchoninic acid Protein assay kit (Wuhan Boster Biological Technology, Ltd.). Protein samples (20 µg) were then separated using 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). The membrane was subsequently blocked with 5% skimmed milk powder for 1 h at room temperature and incubated with primary anti-nuclear factor (NF)-κBp65 (cat. no. 8242) and β-actin (cat. no. 4970) antibodies (Cell Signaling Technology, Inc.) at a dilution of 1:1,000 overnight at 4°C. Samples were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using an ECL chemiluminescence kit (EMD Millipore). Protein levels were calculated relative to β-actin and Image-ProPlus software (version 6.0; Media Cybernetics, Inc.) was used for densitometry analysis.
6
Protein Expression Analysis of Pancreatic Cells
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Total protein was extracted from pancreatic tissue and HPDE6C7 cells using a RIPA buffer with 1% phenylmethylsulfonyl fluoride. Total protein concentration was quantified using the Bicinchoninic Acid Protein Assay kit (Wuhan Boster Biological Technology Co., Ltd.). Protein samples (30 µg/well) were transferred onto polyvinylidene fluoride membranes following 10% and 15% SDS-PAGE. Membranes were blocked with 5% skimmed milk for 45 min at room temperature. The membranes were subsequently incubated with primary antibodies, including those for TRAF6 (1:2,000; cat. no. ab33915; Abcam), NLRP3 (1:1,000; cat. no. IMG-6668A; Novus Biologicals, Inc.), caspase-1 (1:1,000; cat. no. ab179515; Abcam), caspase-3 (1:1,000; cat. no. ab14220; Cell Signaling Technology, Inc.) and β-actin (1:5,000; cat. no. ab6276; Abcam) overnight at 4°C. After washing with TBS-0.1% Tween-20 buffer, membranes were incubated with goat anti-rabbit IgG (1:10,000; cat. no. ab4413; Cell Signaling Technology, Inc.) at room temperature for 1 h. Blots were scanned using the Odyssey® Fc Imager system (LI-COR Biosciences), and protein expression was semi-quantified using imageJ software (version 1.4.1; National Institutes of Health) with β-actin as the loading control.
7
Protein Expression Analysis via Western Blot
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Total protein was isolated from cells using radioimmunoprecipitation assay protein lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were measured using a Bicinchoninic Acid Protein Assay kit (Wuhan Boster Biological Technology, Ltd.) according to the manufacturer's protocol. Proteins (20 µg) were separated via 10% SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% skimmed milk for 2 h at 37°C. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-MMP3 (1:4,000; cat. no. 66338-1-Ig; ProteinTech Group, Inc.), anti-caspase-3 (1:1,000; cat. no. 66470-2-Ig; ProteinTech Group, Inc.), anti-p53 (1:2,000; cat. no. 60283-2-Ig; ProteinTech Group, Inc.) and anti-GAPDH (1:10,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.). After washing three times with PBST (0.05% Tween-20 in PBS) the membranes were incubated with a goat anti-mouse IgG (1:10,000; cat. no. 115-035-003; Jackson ImmunoResearch Laboratories, Inc.) secondary antibody at 37°C for 2 h. Following washing three times with PBST, protein bands were visualized using the ECL assay kit (Beyotime Institute of Biotechnology) and chemiluminescence apparatus (Shanghai Tanon Science & Technology Co., Ltd.). Protein expression was semi-quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.)
8
Western Blot Analysis of Protein Expression
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Total protein was extracted from PC cells using RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.), and the protein concentration was quantified using a bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology, Ltd.). Proteins (30 µg per lane) were loaded on a 10% gel and resolved using SDS-PAGE and transferred to PVDF membranes (EMD Millipore). Membranes were blocked using 5% skimmed milk, and subsequently incubated overnight using primary antibodies against target proteins, FOXO3 (cat. no. 10849-1-AP), β-catenin (cat. no. 51067-2-AP), TCF4 (cat. no. 22337-1-AP), E-cadherin (cat. no. 20874-1-AP), N-cadherin (cat. no. 22018-1-AP), vimentin (cat. no. 10366-1-AP) and GAPDH (cat. no. 60004-1-Ig) (all 1:1,000; ProteinTech Group, Inc.) at 4°C. Subsequently, the membranes were washed using TBS-Tween twice, and subsequently the membranes were incubated with horseradish peroxidase-conjugated anti-mouse (cat. no. BA1051) and anti-rabbit (cat. no. BA1055) secondary antibody (all 1:2,500; Wuhan Boster Biological Technology, Ltd.) and signals were visualized using enhanced chemiluminescent reagent (Wuhan Boster Biological Technology, Ltd.). Image Pro-Plus software was used to analyze the expression of protein, while GAPDH was used as a loading control.
9
Western Blot Analysis of ERCC1 Protein
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Total protein was isolated from transfected cells (5×105 cells/well using radioimmunoprecipitation assay protein lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were measured using a Bicinchoninic Acid Protein Assay kit (Wuhan Boster Biological Technology, Ltd.) following the manufacturer's protocol. Protein samples (20 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk for 2 h at 37°C, the membranes were incubated with anti-ERCC1 antibody (1:2,000; cat. no. 14586-1-AP; ProteinTech Group, Inc.) and anti-β-actin antibody (1:10,000; cat. no. 66009-1-Ig; ProteinTech Group, Inc.) overnight at 4°C. After washing 3 times with PBST (0.05% Tween-20 in PBS), the membranes were incubated with goat anti-rabbit mouse IgG (1:10,000; cat. no. 115-035-003; Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 2 h. After 3 washes, protein bands were visualized using the ECL assay kit (Beyotime Institute of Biotechnology) and analyzed using Image-Pro Plus software v.6.0, (Media Cybernetics Inc.).
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