After indicated treatments, cells were lysed in RIPA buffer containing protease inhibitor. BCA Assay (Applygen Technologies, Beijing, China) was employed to determine protein concentrations. The protein samples were then electrophoretically separated by a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. Then the membrane was blocked in 5% skimmed milk for 1 hour at room temperature. The desired primary antibodies of β-actin (ZSGB-Bio, 1:1000), TrxR1 (Abcam, 1:1800), CD44 (ABGENT, 1:1800), CK7 (Proteintech, 1:2000), E-Cadherin (BioLegend, 1:800), N-Cadherin (Biolegen, 1:800), Oct4 (Bioworld, 1:500), Sox2 (Bioworld, 1:1000), c-Myc (Bioworld, 1:500) and EGFR (ABGENT, 1:2000) were used to incubate with the membranes overnight at 4 °C. The membranes were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) for 1 hour at room temperature. Blots were visualized using the ECL kit (Advansta, Menlo Park, CA, USA). Band intensity was analyzed by Image Lab software, version 4.1 (Bio-Rad Laboratories) and β-actin was used as internal control for normalization.
Bca assay
Manufactured by Applygen
Sourced in China
The BCA assay is a colorimetric detection method used to quantify the total protein concentration in a sample. It is a robust and reliable technique that employs bicinchoninic acid (BCA) to detect and measure the presence of protein.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Merck Group (2 mentions)
Imagelab software version 4, by Bio-Rad (1 mentions)
Ecl kit, by Advansta (1 mentions)
C myc, by Bioworld Technology (1 mentions)
β actin, by Abcam (1 mentions)
β actin, by Bio-Rad (1 mentions)
Trxr1, by Abcam (1 mentions)
E cadherin, by BioLegend (1 mentions)
Colorimetric assay kit, by Keygen Biotech (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Protease inhibitors complete mini, by Roche (1 mentions)
Rabbit anti integrin α5, by Abcam (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Rabbit anti p fak, by Abcam (1 mentions)
Rabbit anti ve cadherin, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Applygen (1 mentions)
Glucose assay kit, by Applygen (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
15 protocols using bca assay
1
Western Blot Analysis of Cellular Proteins
2
Apoptosis Induction by EG-Se/Pt
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Following treatment with EG-Se/Pt, samples containing 5×106 cells were lysed in RIPA lysis buffer (Applygen Technologies, Inc.) on ice for 1 h. The protein concentration was measured by BCA assay (Applygen Technologies, Inc.) and 150 µg protein samples were used for the determination of caspase-3 and caspase-9 activity measured using a Colorimetric Assay kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. The activity of caspase-3 or caspase-9 was determined as the optical density at 405 nm (OD405) in the experimental group/OD405 in the control group using a microplate reader.
3
Oxidative Stress Markers in Kidneys
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Kidneys were homogenized in PBS using a Tissuelyser-38 (Jingxin industrial development co. Ltd, Shanghai, China) and then centrifuged at 3000g for 10 min. The pellet was discarded and protein content was measured in supernatant using the bicinchoninic acid (BCA) assay (Applygen Technologies Inc. Beijing, China). Supernatant aliquots were used to determine the oxidative markers, including nitric oxide (NO), glutathione (GSH), lipid peroxidation (LPO), activity of superoxide dismutase (SOD), and catalase (CAT). The test procedure was followed as specifications of each commercial kits (Nanjing Jiancheng biotechnology, Nanjing, China).
4
Investigating Protein Signaling in RCAECs
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RCAECs (1 × 105/well) were added into the 6-well plates, which were employed for western blotting. A radioimmunoprecipitation assay (RIPA) (Applygen, Beijing, China) involving protease inhibitors (complete mini, Roche) was utilized for extracting the proteins. The BCA assay (Applygen) was employed for estimating the levels of proteins. After protein isolation on SDS-PAGE, they were shifted to nitrocellulose membranes (Millipore). Membranes were blocked with 5% nonfat dry milk for 2 h at room temperature and incubated overnight at 4°C in 5% milk/tris-buffered saline containing 0.1% Tween-20 (TBST) with the following primary antibodies: rabbit anti-p-ephrinB (1 : 1000, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Nck-2 (1 : 1000, Abcam), rabbit anti-p-FAK (1 : 500, Abcam), rabbit anti-VE-cadherin (1 : 1000, Abcam), rabbit anti-Integrinα 5 (1 : 1000, Abcam), and rabbit anti-GAPDH (1 : 2000, Cell Signaling Technology). The membranes were incubated with appropriate peroxidase-conjugated secondary antibodies at 1 : 2000 for 2 h at room temperature, followed by the visualization of immunoreactive proteins on the film using an ECL kit (Millipore, Temecula, CA, USA).
5
Western Blot Protein Analysis Protocol
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Cells were lysed in high salt lysis buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris [pH 8.0], and 5 mM EDTA) with protease inhibitors (10 μg/ml phenylmethylsulfonyl fluoride [PMSF]). After quantification with a BCA assay (Applygen Technologies Inc., Beijing, China), 50 μg total proteins were loaded and separated on SDS-PAGE gel, transferred to PVDF membrane, blocked with 5% non-fat milk, probed with the specified primary antibody, followed by HRP-conjugated secondary antibody, and then visualized with enhanced chemiluminescence (Pierce Supersignal) and X-ray film using standard techniques.
6
Glucose Assay and Protein Quantification
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The glucose content of the medium treated as described above was measured using a glucose assay kit (Applygen Technologies Inc.). The cells were collected and lysed, and the total protein concentration was determined by BCA assay (Applygen Technologies Inc.) to correct for the cell counts.
7
Protein Expression Analysis of eIF5A2 and Doxorubicin
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Cells were harvested in cell lysis buffer (Cell Signaling Technology, Inc.) containing protease inhibitors (Sigma-Aldrich; Merck KGaA) following treatment with eIF5A2 siRNA and doxorubicin for 48 h. The protein concentration was determined by BCA assay (Applygen Technologies, Inc.). A total of 10 µg protein was loaded per lane, separated by 15% SDS-PAGE and transferred to 0.45-µm polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked with TBS-Tween-20 (0.1% Tween-20) containing 0.5% BSA (cat. no. 4240GR100; NeoFroxx GmbH) and incubated with the aforementioned primary antibodies at 4°C overnight. The membranes were washed with TBS-0.1% Tween-20 three times. Following incubation with anti-mouse IgG or anti-rabbit IgG secondary antibodies at 4°C for 2 h, protein expression was detected by chemiluminescence with an EZ-ECL Chemiluminescence Detection Kit (Biological Industries).
8
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer and centrifuged at 12000×g for 5 min at 4°C. The supernatant was collected and the protein concentration was quantified by BCA assay (Applygen Technologies Inc., Beijing, China). Proteins (20 μg) were separated by SDS-PAGE, and then transferred onto nitrocellulose membranes. The membranes were incubated with anti-E-Tmod41 antibody (prepared by AbMax Biotechnology Co., Ltd, Beijing, China), anti-glycophorin A antibody (Beijing Biosynthesis Biotechnology Co. Ltd, Beijing, China), anti-GAPDH antibody, or anti-β-tubulin (Santa cruz Biotech., USA), followed by HRP-conjugated goat anti-rabbit or mouse IgG. The signals were detected by using an Enhanced Chemiluminescence Detection (ECL) kit (Evergreen, Beijing, China).
9
Quantification of Akt and Erk1/2 Phosphorylation in Rat Heart
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Protein was isolated from homogenized heart tissue (n = 4) with nondenaturing lysis buffer added with proteinase inhabitor reagent and the concentrations were calculated by BCA assay(Applygen Technologies Inc., Beijing, China). To determine the phosphorylated level ofAkt(serine[Ser] 473) and Erk1/2 in addition to the total level of the proteins(Cell SignalingTechnology, Beverly, MA), 40 μg homogenized protein was separated on a Bis-Tris Gel and transferred to a nitrocellulose mini membrane (Invitrogen, Carlsbad, CA). Membranes were then probed with a rabbit polyclonal antibody against rat Akt(serine[Ser] 473) and Erk1/2(Cell Signaling Technology, Beverly, MA). The bound primary antibodies were detected with horseradishperoxidase-conjugated secondary antibodies and enhanced chemi- luminescencedetection reagents (Applygen Technologies Inc., Beijing, China). The reported densitometry values were analyzed by Totalab v1.10 software (Totalab Ltd., Newcastle, U.K.) and normalized to total Akt and Erk1/2.
10
Kidney Oxidative Stress Markers Assay
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Kidneys were homogenized in Phosphate-buffered saline (PBS) using a TissueLyser-38 (Jingxin Industrial Development Co. Ltd., Shanghai, China) and then centrifuged at 3,000 g for 10 min. The pellet was discarded and protein content was measured in the supernatant using a bicinchoninic acid (BCA) assay (Applygen Technologies Inc., Beijing, China). Supernatant aliquots were used to determine oxidative markers, including nitric oxide (NO), reduced glutathione (GSH), lipid peroxidation (LPO), superoxide dismutase (SOD) activity, and catalase (CAT) activity. The test procedure supplied with each commercial kit was followed (Nanjing Jiancheng Biotechnology, Nanjing, China).
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