Total protein was extracted from tissues or cells using cold radioimmunoprecipitation assay buffer (Nanjing KeyGen Biotech Co., Ltd, Nanjing, China), and the concentration of total protein was measured using a bicinchoninic acid assay kit (Nanjing KeyGen Biotech Co., Ltd). Equal amounts of protein were separated on 10% sodium dodecyl sulfate polyacrylamide gel, transferred onto polyvinylidene fluoride membranes and blocked at room temperature for 2 h with Tris-buffered saline containing 0.1% Tween-20 (TBST) containing 5% fat-free milk. Next, the membranes were incubated overnight at 4°C with primary antibodies against HMGA2 (1:1,000 dilution; cat. no. ab97276; Abcam, Cambridge, UK) or GAPDH (1:1,000 dilution; cat. no. ab128915; Abcam). Following a wash with TBST, membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated second antibody (1:5,000 dilution; cat. no. ab6721; Abcam) at room temperature for 2 h. The membranes were then washed again with TBST. Protein bands were visualized using enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.). GAPDH was used as an internal control for HMGA2 protein expression.
Bicinchoninic acid assay kit
Manufactured by Keygen Biotech
Sourced in China
The Bicinchoninic acid (BCA) assay kit is a colorimetric detection method used for the quantitative determination of total protein concentration in a sample. The kit contains reagents necessary for performing the assay, including the bicinchoninic acid solution and a copper (II) sulfate solution. The BCA assay is based on the reduction of Cu2+ to Cu+ by proteins in an alkaline medium, with the purple-colored reaction product being formed by the chelation of two molecules of BCA with one cuprous ion.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence kit, by Cytiva (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Image pro plus 6.0 analysis system, by Media Cybernetics (2 mentions)
Horseradish peroxidase labeled secondary antibody igg, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Radioimmunoprecipitation assay buffer, by Keygen Biotech (1 mentions)
Chemiluminescent horseradish peroxidase substrate, by Merck Group (1 mentions)
Anti rock1, by Cell Signaling Technology (1 mentions)
Anti p mypt1, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody, by Proteintech (1 mentions)
Tunel in situ cell death detection kit, by Roche (1 mentions)
3 methyladenine 3 ma, by Selleck Chemicals (1 mentions)
Cell cycle detection kit, by Keygen Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (1 mentions)
Cycloheximide (chx), by MedChemExpress (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
10 protocols using bicinchoninic acid assay kit
1
Western Blot Analysis of HMGA2 Protein
2
Quantification of Platelet Protein Levels
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Total protein from the platelets was extracted using radioimmunoprecipitation assay lysis buffer combined with phenylmethanesulfonyl fluoride and phosphatase inhibitors and quantified using a bicinchoninic acid assay kit (KeyGEN BioTECH). Protein samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then semi-wet transferred onto polyvinylidene fluoride membranes. Non-specific binding sites were blocked with 5% BSA dissolved in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 hour at room temperature. Membranes were respectively incubated with primary antibodies of anti-ROCK1, anti-caspase-3, anti-myosine phosphatae targeting subunit 1 (MYPT1), anti-p-MYPT1 (Cell Signaling Technology), and anti-GAPDH (Proteintech Group) overnight at 4°C. Peroxidase-conjugated secondary antibodies were incubated with the membranes for 1 hour. Detection of the bands was performed using chemiluminescent horseradish peroxidase substrate (Merck Millipore) with an ImageQuant LAS 4000 mini acquisition system.
3
Apoptosis, Autophagy, and Oxidative Stress Analysis
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Annexin V-enhanced green fluorescent protein (EGFP)/propidium iodide (PI) apoptosis detection kit, cell cycle detection kit, DAPI, bicinchoninic acid assay kit, and cell lysis assay kit containing protease and phosphatase inhibitors were purchased from Nanjing Keygen Biotech, Co., Ltd. 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and trypan blue dye were purchased from Beyotime Institute of Biotechnology. In situ cell death detection TUNEL kit was purchased from Roche Diagnostics GmbH. 3-Methyladenine (3-MA) and rapamycin were purchased from Selleck Chemicals. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (Merck KGaA). Cycloheximide (CHX) was purchased from MedChem Express. Rabbit monoclonal antibodies against β-actin (cat. no. 4970), γ-H2AX (cat. no. 9718), caspase-3 (cat. no. 9662), cleaved caspase-3 (cat. no. 9664), LC3 (cat. no. 3868), CHOP (cat. no. 5554) and Ki-67 (cat. no. 9027) were obtained from Cell Signaling Technology, Inc. Rabbit polyclonal antibodies against p62 (cat. no. 18420) and Grp78/Bip (cat. no. 11587) were obtained from ProteinTech Group, Inc. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. G-21234) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (cat. no. A-11008) were obtained from Invitrogen (Thermo Fisher Scientific, Inc.).
4
Protein Expression Analysis in MC-4 Cells
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MC-4 cells were collected following treatment with pPeOp (30, 60 or 90 µg/ml), 90 µg/ml PVP or 100 µg/ml 5-FU, subsequently lysed with radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology) for 30 min on ice. The lysates were separated by centrifugation at 12,000 × g for 15 min at 4°C. The total protein concentration in the supernatants was determined using a bicinchoninic acid assay kit (Nanjing KeyGen Biotech Co., Ltd., Nangjing, China), according to the manufacturer's protocol. SDS-PAGE was performed using 10% gels with ~20 µg protein per lane. Proteins were subsequently transferred onto polyvinylidene fluoride membranes, which were blocked with 5% dried skimmed milk in Tris-buffered saline containing 1% Tween-20 (TBST) and incubated with primary antibodies (described above) overnight at 4°C. Membranes were washed three times with TBST and incubated with HRP-conjugated goat anti-rabbit IgG at room temperature for 2 h, followed by washing three times with TBST. Images of the blots were captured on film in a darkroom using BeyoECL Plus Substrate kit (Beyotime Institute of Biotechnology) and were scanned and quantified using the Quantity One 1-D image analysis software (version 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
5
Western Blot Analysis of Apoptosis and NF-κB Signaling
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Cells were lysed using RIPA buffer and total protein was extracted from the lysate. Total protein concentration was determined using a bicinchoninic acid assay kit (KeyGen, Nanjing, China). Protein samples were separated by electrophoresis on a 13% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk at 25 °C for 1 h, the membrane was incubated with the following primary antibodies: anti-Bcl-2 (1:1000; Abcam, USA), anti-Bax (1:1000; Abcam), anti-P-Iκnt (1:1000; Beyotime, China), anti-Iκnt (1:1000; Beyotime, China), anti-P-p65 (1:1000; Abcam), anti-p65 (1:1000; Abcam), and anti-β-actin (1:500; Beyotime, Hangzhou, China) overnight at 4 °C. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated with horseradish peroxidase-labeled secondary antibody IgG (1:1000; Abcam, USA) for 1 h at 37 °C. Blots were visualized with an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) and quantified using the Image Pro-Plus 6.0 analysis system (Media Cybernetics, Rockville, MD, USA). Band density values were normalized to β-actin.
6
Quantification of FASN Protein Levels
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A total of three GC tissues were lysed and subjected to western blotting as described previously (25 (link)). Briefly, the protein concentrations of the lysates were quantified using a Bicinchoninic Acid Assay kit (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China). Protein samples (40 µg) were separated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Shanghai Shenggong Biology Engineering Technology Service, Ltd., Shanghai, China) and were then electrophoretically transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were incubated in 5% bovine serum albumin (ZSGB-BIO, Beijing, China) in PBS for 1 h, then with rabbit anti-human FASN polyclonal antibodies (1:1,000; 3180S; Cell Signaling Technology, Inc.), overnight at 4°C. Membranes were then washed three times with PBS and incubated with HRP-conjugated goat anti-rabbit IgG (1:15,000; PV-6001; LI-COR, Inc.) for 1 h at room temperature. Antibody complexes were detected using enhanced chemiluminescence (PerkinElmer, Inc., Waltham, MA, USA) and the blots were scanned using the Odyssey CLx Imaging System (LI-COR, Inc.). The intensity of protein bands were quantified using Quantity One software, version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
7
Protein Expression Analysis in Intestinal Cells
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The intestinal tissues and Caco-2 cells were homogenized and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (keygen BioTECH, Nanjing, China), supplemented with 1% phenylmethylsulfonyl fluoride (PMSF) (keygen BioTECH). The concentration of the extracted proteins was determined using the Bicinchoninic acid assay kit (keygen BioTECH). Protein solutions were mixed with sodium dodecyl sulfate (SDS) sample buffer (keygen BioTECH) in a 4:1 ratio and denatured in boiling water for 10 min. Protein samples were separated on a 10% polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was blocked in rapid blocking solution at room temperature for 10 min, followed by overnight incubation at 4 °C with antibodies against hydroxycarboxylic acid receptor 2 (Hcar2) (Affinity Biosciences, Jiangsu, China), claudin3 (Affinity Biosciences), claudin4 (ZENBIO Biotechnology, Chengdu, China), β-actin (ABclonal, Wuhan, China), and β-Tubulin (ZENBIO Biotechnology). Subsequently, the membrane was incubated with a labeled secondary anti-rabbit antibody for 1 h, and the immunoreactive protein bands were detected using an enhanced chemiluminescence (ECL) kit (Millipore) and visualized using the Bio-Rad ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA). Image analysis was performed using Image Lab and ImageJ software.
8
Resveratrol-induced autophagy evaluation
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Cells were treated with 40 µM Res for 24 or 48 h. For some experiments, 10 mM NAC, 20 µM SP600125 or 20 µM SB203580 were used. In order to evaluate autophagic flux, cells were treated with 40 µM Res for 48 h in the presence or absence of 50 µM CQ.
Cells were lysed using a total protein extraction kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's protocol. Protein concentrations were determined using a bicinchoninic acid assay kit (Nanjing KeyGen Biotech Co., Ltd.) and 30 µg protein/lane was separated via SDS-PAGE on a 10–12% gel. The separated proteins were subsequently transferred onto a polyvinylidene difluoride membrane and blocked with 5% BSA 1 h at room temperature. The membranes were incubated with the aforementioned primary antibodies, overnight at 4°C. The membranes were then washed three times with TBST and incubated with corresponding anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:5,000; catalog no. GAR007 and GAM007; MultiSciences Biotech, Co., Ltd.) at room temperature for 1 h. Protein bands were visualized using an ECL system (EMD Millipore) and densitometric analysis was performed using ImageJ software (version 1.48v; National Institutes of Health).
Cells were lysed using a total protein extraction kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's protocol. Protein concentrations were determined using a bicinchoninic acid assay kit (Nanjing KeyGen Biotech Co., Ltd.) and 30 µg protein/lane was separated via SDS-PAGE on a 10–12% gel. The separated proteins were subsequently transferred onto a polyvinylidene difluoride membrane and blocked with 5% BSA 1 h at room temperature. The membranes were incubated with the aforementioned primary antibodies, overnight at 4°C. The membranes were then washed three times with TBST and incubated with corresponding anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (1:5,000; catalog no. GAR007 and GAM007; MultiSciences Biotech, Co., Ltd.) at room temperature for 1 h. Protein bands were visualized using an ECL system (EMD Millipore) and densitometric analysis was performed using ImageJ software (version 1.48v; National Institutes of Health).
9
Western Blot Analysis of Hypoxia Markers
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Cells were lysed using RIPA buffer and total protein was extracted from the lysate. Total protein concentration was determined using a bicinchoninic acid assay kit (KeyGen, Nanjing, China). Protein samples were separated by electrophoresis on a 13% sodium dodecyl sulfate–polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk at 25°C for 1 h, the membrane was incubated with the following primary antibodies: anti‐HIF‐1α (1:1000; Abcam, UK), anti‐FIH‐1 (1:1000; Abcam), anti‐VEGF (1:1000; Beyotime, China), anti‐GAPHD (1:1000; Beyotime, China). After washing with Tris‐buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated with horseradish peroxidase‐labeled secondary antibody IgG (1:1000; Abcam, UK) for 1 h at 37 °C. Blots were visualized with an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) and quantified using the Image Pro‐Plus 6.0 analysis system (Media Cybernetics, Rockville, MD, USA). Band density values were normalized to GAPDH.
10
Quantitative Protein Analysis of Liver Tissue
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For western blot analysis, liver tissue samples were homogenized in 1 mL of lysis buffer (KeyGEN BioTECH, China) and protein concentrations were quantified using a bicinchoninic acid assay kit (KeyGEN BioTECH). Equal amounts of total protein (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Temecula, CA, USA). The membranes were blocked with 5% BSA for 1 h and then incubated with primary antibodies at 4°C overnight. Anti-GAPDH and anti-beta-actin antibodies were used as internal controls. The antibodies used in this study are listed in Table S8 .
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