Ice-cold lysis buffer (Beyotime) was utilized to lyse HPAEpiCs. The proteins were separated and transferred onto polyvinylidene fluoride membrane, which was blocked in nonfat dried milk. Subsequently, the polyvinylidene fluoride membranes were incubated with the following primary antibodies: anti-inducible NOS (anti-iNOS; Cat#MA5-17139; 1:1000; Thermo Fisher, Waltham, MA, USA), anti-cyclooxygenase-2 (anti-COX2; Cat#MA5-14568; 1:1000; Thermo Fisher), anti-p-p65 (Cat#AF2006; 1:2000; Affinity, Nanjing, China), anti-p65 (Cat#AF5006; 1:1000; Affinity) and anti-GAPDH (Cat#MA1-16757; 1:1000; Thermo Fisher). After being washed with PBS, secondary antibodies (Cat#A32731/A32723) were used to incubate the membranes. At last, immunoreactive bands were visualized using eyoECL Plus (Beyotime).
Eyoecl plus
Manufactured by Beyotime
Sourced in China
The EyoECL Plus is a laboratory equipment used for electrochemiluminescence (ECL) detection. It provides a sensitive and reliable method for analyzing and quantifying various biomolecules, such as proteins, nucleic acids, and small molecules, in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
B cell lymphoma 2 bcl 2, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Skimmed milk, by Solarbio (2 mentions)
Np 40 buffer, by Beyotime (2 mentions)
Ice cold lysis buffer, by Beyotime (1 mentions)
Anti p65, by Affinity Biosciences (1 mentions)
Anti p p65, by Affinity Biosciences (1 mentions)
Anti gapdh, by Thermo Fisher Scientific (1 mentions)
C myc, by Thermo Fisher Scientific (1 mentions)
Cyclin d1, by Affinity Biosciences (1 mentions)
β catenin, by Affinity Biosciences (1 mentions)
Mmp 9, by Abcam (1 mentions)
Proliferating cell nuclear antigen (pcna), by Thermo Fisher Scientific (1 mentions)
Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions)
Surepage gels, by Beyotime (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Loading buffer, by Thermo Fisher Scientific (1 mentions)
9 protocols using eyoecl plus
1
Western Blot Analysis of Inflammatory Markers
2
Western Blot Analysis of Protein Expression
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With respect to the detection of protein expression, Western blot was carried out as previously described [24] (link). After 48 h of transfection, cells were harvested for analysis of protein expression. In brief, a total of 20 μg protein extract from tissues and cells was loaded onto polyacrylamide gels and transferred onto nitrocellulose membranes prior to blocking aspecific signals using defatted dry milk. Then, the membranes were incubated with the antibodies of proliferating cell nuclear antigen (PCNA; 1:1000; Thermo Fisher), B-cell lymphoma 2 (Bcl-2; 1:500; Thermo Fisher), Bcl-2-like protein 4 (Bax; 1:6000; Thermo Fisher), TRIM66 (1:1000; Thermo Fisher), β-catenin (1:2000; Affinity, Nanjing, China), c-myc (1:1000; Thermo Fisher), cyclinD1 (1:2000; Affinity), MMP2 (1:3000; Abcam, Cambridge, UK) and MMP9 (1:5000; Abcam) and GAPDH (1:400; Thermo Fisher). The blots were probed with secondary antibodies (1:4000; Thermo Fisher). At last, protein visualization was achieved using eyoECL Plus (Beyotime, Shanghai, China).
3
Western Blot Analysis of EMT Markers
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As described by Chen et al and his colleagues [25 (link)], after 48 h of transfection, cells were harvested for protein extraction. Proteins were quantified with a BCA protein assay kit (Solarbio). Then, an aliquot of 20 μg protein was electrophoresed on SurePAGE gels (Beyotime) and transferred onto nitrocellulose membranes using XCell II Blot Module (Thermo Fisher). The membranes were subjected to incubation with the antibodies for N-cadherin (1:500; Thermo Fisher), E-cadherin (1:1000; Thermo Fisher), SPARCL1 (1:1000; Thermo Fisher), and β-actin (1:3000; Affinity, Nanjing, China) after being blocked with 5% skimmed milk (Solarbio). The nitrocellulose membranes were incubated with secondary antibodies (1:3000; Affinity). At last, a Bio-Rad image analysis system was performed to develop protein blots with eyoECL Plus (Beyotime). The protein expression was normalized to β-actin.
4
Western Blot Analysis of Apoptosis Markers
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NP-40 buffer (Beyotime, Shanghai, China) was firstly employed to lyse cells, and lysates were mixed with loading buffer (Thermo Fisher). The mixture was then boiled in boiling water. After that, lysates were loaded by 15% bis-tris-acrylamide gel (Thermo Fisher). The protein bands were electrotransferred onto nitrocellulose membranes (Membrane Solutions, Shanghai, China), which were then immersed in 8% defatted milk (Solarbio). Then, the membranes were incubated with primary antibodies against B-cell lymphoma-2 (Bcl-2) (1:1000; Abcam, Cambridge, UK), BCL2-associated x protein (Bax) (1:5000; Abcam), Cleaved caspase 3 (c-caspase 3; 1:5000; Abcam), pro-caspase 3 (p-caspase 3; 1:2000; Abcam), BRD4 (1:800; Abcam) and β-actin (1:1000; Abcam), respectively. Afterwards, secondary antibody against IgG H&L (Abcam; 1:8000) was utilized to incubate the membranes. Finally, the protein bands were visualized by dropwise adding eyoECL Plus (Beyotime) onto the membranes. β-actin was employed as a control.
5
Protein Extraction and Western Blot Analysis
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Concisely, cells and tissues were incubated with Protein Extraction Reagent (Phygene, Fuzhou, China) containing Protease Inhibitor Cocktail for 20 min on ice, and total proteins were extracted by centrifugation at 10,000g for 20 min. Then, 20 μg of protein were loaded onto 8%-12% bis–tris-acrylamide gels (Phygene). The proteins were wet-transferred onto nitrocellulose membranes, which were then blocked with 5% defatted dry milk. Next, the primary antibodies specific to BCL2-associated x protein (Bax; 1:1000; Thermo Fisher), cleaved caspase 3 (1:1500; Thermo Fisher), B-cell lymphoma-2 (Bcl 2; 1:50; Thermo Fisher), HDAC4 (1:1500; Thermo Fisher) and GAPDH (1:1000; Thermo Fisher) were used to incubate the membranes. After being probed with secondary antibodies (Thermo Fisher), the membranes were analyzed under a Bio-Rad image analysis system with the use of eyoECL Plus (Beyotime).
6
Western Blot Analysis of Metalloproteinases
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The tissue and cell samples were lysed using NP-40 buffer (Beyotime), and proteins were degenerated by heating. Then, an aliquot of 20 μg protein samples were electrophoresed on 10% bis–tris-acrylamide gel (Thermo Fisher), followed by the wet-transfer with the nitrocellulose membranes (Pall Life Science, Beijing, China). After blocking in skimmed milk (Solarbio), the membranes were probed with anti-matrix metalloprotein 9 (anti-MMP9; 1:1000; Affinity, Nanjing, China), anti-MMP2 (1:1000; Affinity), anti-MAP3K3 (1:1000; Affinity) and anti-GAPDH (1:8000; Affinity). After that, the secondary antibody against goat anti-rabbit IgG (1:5000; Affinity) was incubated with the membranes. The protein signals were detected with eyoECL Plus (Beyotime). GAPDH was employed to normalize protein abundance.
7
Protein Extraction and Western Blot Analysis
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Total proteins from the blood samples of AS patients and HVSMCs were isolated using RIPA lysis buffer containing a protease inhibitor (Sbjbio® life science). The concentrations of extracted proteins were determined with a BCA protein assay kit (Sbjbio® life science). Then, polyacrylamide gels were used to separate protein with a SureLock Midi-Cell Electrophoresis System. After blocking the aspecific signals using skimmed milk (Yili, Beijing, China), polyvinylidene fluoride membranes with protein bands were incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA) (#13-3900; 1:5000; Thermo Fisher, Waltham, MA, USA), matrix metalloprotein 2 (MMP2) (#436000; 1:250; Thermo Fisher), IGF2 (#MA5-17096; 1:1,000; Thermo Fisher), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#MA1-16757; 1:2,000; Thermo Fisher). After being exposed to eyoECL Plus (Beyotime, Shanghai, China), the visualized blots were analyzed using Image J software.
8
Proteomics Analysis of Placental Tissues
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Total proteins from placental tissues and cells were extracted using NP-40 lysis buffer (Beyotime). 20 μg protein, as quantified with BCA protein assay kit (Beyotime), was loaded onto bis-tris-acrylamide gels and then transferred onto nitrocellulose membranes. The proteins were detected using the following primary antibodies: anti-N-cadherin (Cat# MA5-15633; 1:1000; Thermo Fisher), anti-E-cadherin (Cat# PA5-32178; 1:5000; Thermo Fisher), anti-RYBP (Cat# PA1-26492; 1:1000; Thermo Fisher), and anti-β-actin (Cat# PA5-16914; 1:500; Thermo Fisher) and secondary antibodies (Thermo Fisher). At last, protein visualization was performed using eyoECL Plus (Beyotime).
9
Protein Analysis in Cancer Cell Lines
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The protein levels in A549 and H1975 cells were assessed by western blot assay.
Cells from different groups were collected and lysed in RIPA buffer (Beyotime, China) containing 1% PMSF (Beyotime) on ice for 5 min. After centrifugation at 10,000 g for 10 min at 4°C, the cell supernatants were collected. Protein concentration was measured using an Enhanced BCA Protein Assay Kit (Beyotime). Protein samples (40 μg) were subjected onto SDS-PAGE and blotted to PVDF membranes (Millipore, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with primary antibodies LC3I/II (1:1000,Cell Signaling Technology),Cell Signaling Technology),Cell Signaling Technology),JNK1/2 (1:1000,Sangon,China),Bioss,China) at 4°C overnight. Then, the membranes were incubated with HRP-labeled Goat Anti-Rabbit IgG(H+L) (1:5000, Beyotime) at 37°C for 45 min. Blots were visualized using eyoECL Plus (Beyotime) and analyzed by Gel-Pro-Analyzer software.
Cells from different groups were collected and lysed in RIPA buffer (Beyotime, China) containing 1% PMSF (Beyotime) on ice for 5 min. After centrifugation at 10,000 g for 10 min at 4°C, the cell supernatants were collected. Protein concentration was measured using an Enhanced BCA Protein Assay Kit (Beyotime). Protein samples (40 μg) were subjected onto SDS-PAGE and blotted to PVDF membranes (Millipore, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with primary antibodies LC3I/II (1:1000,Cell Signaling Technology),Cell Signaling Technology),Cell Signaling Technology),JNK1/2 (1:1000,Sangon,China),Bioss,China) at 4°C overnight. Then, the membranes were incubated with HRP-labeled Goat Anti-Rabbit IgG(H+L) (1:5000, Beyotime) at 37°C for 45 min. Blots were visualized using eyoECL Plus (Beyotime) and analyzed by Gel-Pro-Analyzer software.
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