Total cell proteins were extracted in RIPA lysis buffer. The protein concentration was determined by using BCA method (Biyuntian, Jiangsu, China). Equal amount of proteins were loaded on a 10 % SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel. Following electrophoresis, the proteins were blotted to a PVDF membrane. The membrane was blocked in 5 % non-fat milk and then incubated with primary antibodies overnight at 4 °C and secondary antibody reactions for 2 h at 37 °C. Sources of primary antibodies were: anti-SALL4, anti-Vimentin and anti-E-cadherin (Abcam, Cambridge, MA, USA), anti-β-catenin, anti-wnt3a, anti-β-actin antibodies (Cell Signaling Technology, USA)
Bca method
Manufactured by Beyotime
Sourced in China
The BCA method is a colorimetric assay used for the quantitative determination of protein concentration. It utilizes the reduction of copper ions (Cu2+) to cuprous ions (Cu1+) by protein in an alkaline environment, and the subsequent chelation of the cuprous ions with bicinchoninic acid to produce a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (70 mentions)
Ripa buffer, by Beyotime (32 mentions)
Ripa lysis buffer, by Beyotime (30 mentions)
Odyssey infrared imaging system, by LI COR (8 mentions)
Gapdh, by Cell Signaling Technology (7 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (7 mentions)
Pvdf membrane, by Thermo Fisher Scientific (7 mentions)
Gapdh, by Abcam (6 mentions)
Ab32503, by Abcam (6 mentions)
Ab9485, by Abcam (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (6 mentions)
Sds page gel, by Beyotime (6 mentions)
β actin, by Abcam (5 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (5 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
Ripa lysis buffer, by Merck Group (5 mentions)
Anti β actin, by Abcam (5 mentions)
Pvdf membrane, by Roche (5 mentions)
Anti e cadherin, by Abcam (4 mentions)
289 protocols using bca method
1
Western Blot Analysis of Cell Proteins
2
Protein Extraction from Liver Tissue
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Fresh liver tissue (100 mg) was taken and ground using liquid nitrogen. The total protein content was extracted from the liver tissues using a total protein extraction kit containing protease inhibitors (Biyuntian Biotechnology Co., Ltd., China) and phosphatase inhibitors (Biyuntian Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. The protein concentration was determined by the BCA method (Biyuntian Biotechnology Co., Ltd., China). Total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electropheresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk in Tris-buffered saline (1x) for 2 h, the membranes were probed with appropriate primary antibodies at 4°C overnight and then detected by HRP-labeled anti-rabbit or HRP-labeled anti-mouse secondary antibodies (Zhongshan Jinqiao Biotechnology Co, Ltd., China). The antigen-antibody complex was detected by an enhanced ECL reagent (Biyuntian Biotechnology Co., Ltd., China). After exposure, Image J was used for grayscale analysis.
3
Western Blot Analysis of Protein Expression
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Total protein from Huh-7 cells was extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology) and detected via the BCA method (Beyotime Institute of Biotechnology). Equal quantities (30 µg) of protein were electrophoresed on 6–12% denaturing SDS gels at 110 V for 90 min, transferred to PVDF membranes (EMD Millipore) and blocked for 1 h at room temperature. The membranes were then incubated with 1% BSA (11021045; Invitrogen; Thermo Fisher Scientific), followed by incubation with antibodies against CCL23 (1:2,000; PA5-100686; Invitrogen; Thermo Fisher Scientific), TFAP4 (1:1,000; HPA001912; Sigma-Aldrich; Merck KGaA), matrix metalloproteinase-2 (MMP2) (1:3,000; sc-13594; Santa Cruz Biotechnology), MMP9 (1:3,000; ABT544; EMD Millipore) and GAPDH (1:5,000; 5174; Cell Signaling Technology) overnight at 4°C. After washing with TBST at room temperature for 30 min, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibodies (1:1,000; 7076, 7074; Cell Signaling Technology) for 2 h at room temperature. The antigen–antibody complexes were visualized with the ECL system (WBULS0500; EMD Millipore). The ImageJ 1.4.3 was used for densitometry analysis of the protein bands [25 (link)].
4
Western Blot Protein Analysis
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Total cell proteins were extracted from lysed cells with RIPA lysis buffer (Beyotime). The protein concentration was tested with BCA method (Beyotime). A total of 40 µg of protein was separated by 10% SDS-polyacrylamide gels, which was then transferred onto nitrocellulose membranes (Merck KGaA). After blocking with 5% skimmed milk for 1.5 h, the membranes were first probed with specific primary antibodies at 4 °C overnight. Subsequently, these blots were incubated with peroxidase-conjugated secondary antibody for 1.5 h at room temperature. The proteins were finally visualized under enhanced chemiluminescence (EMD Millipore). Band densities of target proteins were normalized to that of GAPDH and quantified using Image J software.
5
Western Blot Analysis of Apoptosis and Integrin Signaling
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Total protein was extracted with ice-cold RIPA buffer (Thermo Fisher Scientific) and then quantified using BCA method (Beyotime). Proteins (30 μg per lane) were loaded onto 10% SDS-PAGE. Then, the proteins were electro-transferred onto polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific) and blocked in 5% skimmed milk for 1 h. Later on, the membrane was incubated in primary antibodies at 4°C overnight. Rabbit against Bcl-2 (1:1000, Abcam), cleaved caspase 3 (1:1000, Abcam), RSU1 (1:1000, Abcam), PINCH1 (1:1000, Abcam), ILK (1:1000, Abcam) and β-actin (1:1000, Abcam). After that, anti-rabbit IgG HRP-conjugate secondary antibodies (Abcam) were reacted with the membrane at room temperature for 1 h. Subsequently, the membrane was visualized using an enhanced chemiluminescence (Thermo Fisher Scientific). β-actin was acted as the internal control.
6
Hippocampal Protein Quantification and Western Blot
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Each group of mice was decapitated immediately after the electrophysiological experiment. The entire brain was removed from the skull and the hippocampus (n = 3 mice per group) was extracted carefully. The hippocampus was harvested and lysed on ice for 15 min in 150 mL lysis buffer. The tissue homogenate was centrifuged at 12,000 rpm and 4°C for 30 min.
The protein concentration in the supernatant was quantified by the BCA method (Beyotime Biotechnology, Haimen, China). The supernatant was mixed with 5× loading buffer and boiled at 100°C for 15 min to denature the protein. Equivalent amounts of protein (30 μg) were run using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8%-13% gels and then transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated with Tween-20 (TBST) with 5% skim milk for 1 h at room temperature to block nonspecific protein binding. Subsequently, the membranes were incubated with the primary antibodies overnight at 4°C. After washing with TBST buffer, the PVDF membranes were incubated with secondary antibodies for 50 min at room temperature. A chemiluminescence imaging system was used to image the PVDF membrane and the ImageJ program (National Institutes of Health, Bethesda, MD, United States) was used to analyse greyscale values.
The protein concentration in the supernatant was quantified by the BCA method (Beyotime Biotechnology, Haimen, China). The supernatant was mixed with 5× loading buffer and boiled at 100°C for 15 min to denature the protein. Equivalent amounts of protein (30 μg) were run using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8%-13% gels and then transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated with Tween-20 (TBST) with 5% skim milk for 1 h at room temperature to block nonspecific protein binding. Subsequently, the membranes were incubated with the primary antibodies overnight at 4°C. After washing with TBST buffer, the PVDF membranes were incubated with secondary antibodies for 50 min at room temperature. A chemiluminescence imaging system was used to image the PVDF membrane and the ImageJ program (National Institutes of Health, Bethesda, MD, United States) was used to analyse greyscale values.
7
Western Blot Analysis of Apoptosis and EMT Markers
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MCF7 and MDA-MB-231 cells were seeded in 6-well plates (6×105/well). Seventy-two hours later, the total protein was extracted from the two cell lines using RIPA (Beyotime, Shanghai, China). The BCA method (Beyotime, Shanghai, China) was employed for protein content measurement. 20 μg total protein in each group was subjected to 10% polyacrylamide gel electrophoresis and then transferred to the PVDF membranes, which were blocked with 5% skimmed milk. Afterward, the membranes were incubated with the primary antibodies (Abcam, MA, USA), including anti-Casase3 (ab32351, 1: 1000), anti-Bax (ab32503, 1: 1000), anti-Bcl2 (ab32124, 1: 1000), anti-Vimentin (ab92547, 1: 1000), anti-E-cadherin (ab40772, 1: 1000), anti-N-cadherin (ab76011, 1: 1000), anti-FASN (ab22759, 1: 1000), anti-AMPK (ab3047, 1:1000), anti-p-AMPK (ab23875, 1: 1000), anti-mTOR (ab2732, 1: 1000) and anti-p-mTOR (ab109268, 1:1000) at 4°C overnight. Next, they were kept with the goat anti-rabbit IgG (ab150077, 1: 2500, Abcam, MA, USA) at room temperature (RT) for 2 hours. Subsequently, the ECL chemiluminescence reagent was used for protein blots exposure. ImageJ software was used for gray analysis. GAPDH acted as the internal control.
8
Protein Extraction and Western Blot Analysis of Brain and Cell Samples
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Mouse brain tissue, human brain tissue, and cultured cells (collected after washing with PBS) were each placed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) (RIPA: PMSF = 100:1) for 30 min, homogenized using a tissue grinder, and centrifuged at 10,000 rpm for 20 min at 4°C. The supernatants were collected and total protein concentrations were measured by the BCA method (Beyotime, Shanghai, China). After dilution with loading buffer, proteins were subjected to SDS-PAGE and transferred to polyvinylidene fluoride, which was then blocked with 5% skim milk for 1 h, and probed with different primary antibodies (1:1000, v/v) at room temperature for 1 h. The membranes were then incubated with secondary antibody (1:10,000, v/v) for 1 h at room temperature. The bands were visualized by enhanced chemiluminescence and observed under ChemiDoc imaging instrument (Bio-Rad Laboratories, Shanghai, China). The optical density of the bands was analyzed using Image J software. GAPDH served as an internal control.
9
Neutrophil Activation and APOE3 Effects
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Neutrophils were harvested and divided into 3 subgroups (control, PMA, and PMA+APOE3). After being incubated with PBS, PMA, or PMA plus APOE3 for 1 hour, cells were centrifuged and lysed with RIPA buffer (Beyotime Institute of Biotechnology). Total protein was measured via the BCA method (Beyotime Institute of Biotechnology), and samples were boiled with InstantView™ SDS-PAGE loading buffer (Beyotime Institute of Biotechnology). Samples were separated by 12% SDS-PAGE gel (Beyotime Institute of Biotechnology) and transferred onto a PVDF membrane (Millipore). Total protein bands were visualized by using UV light (ChemiDoc™ Imaging System, Bio-Rad). Membranes were blocked with 5% (wt/vol) milk in TBST for 1 hour, then incubated with primary antibodies (Cell Signaling Technology) overnight. The blots were rinsed with TBST three times and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 40 min. After being washed with TBST three times, the blots were applied to an ECL luminol reagent (Beyotime Institute of Biotechnology) and visualized via the ChemiDoc™ Imaging System. Bands were quantified by the ImageJ software.
10
Protein Expression Analysis in Rat Heart
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The total proteins from rat heart tissues or AC16 cells were extracted by using the Protein Rapid Extraction kit (KeyGEN, China). After protein quantification by the BCA method (Beyotime, China), 30 μg for each sample was used to perform the Western blot assay as described previously. In this study, the primary antibodies for ferritin (heavy chain, FTH1; light chain, FTL), TFRC/TfR, DMT1, ACSL4, GPX4, and SLC7A11 were all purchased from Novus, USA, whilst that for HO-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from CST, USA. Finally, the imaged blots were quantified by dividing the signal of the tested protein by corresponding GAPDH (the loading control) and normalized to the controls.
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