Total protein was extracted using urea-based denaturing buffer (100 mM NaH2PO4, 8 M urea, and 10 mM Tris–HCl, pH 8.0) and used for immunoblot analysis to check the expression. The proteins were fractionated by 8% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). The membrane was treated with 5% nonfat milk in PBS-T for 10 min for blocking, and then incubated with Cas9 antibody (Santa cruz, 7A9-3A3, 1:500) at 4 °C for overnight. After incubation, the membrane was washed three times for 5 min and incubated with horseradish peroxidase-conjugated anti-mouse (1:10,000) for 2 h. The Blot was washed with PBS-T three times and detected with the ECL system (Thermo scientific, lot# SE251206).
Ecl system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark, China, United Kingdom, Germany, Japan
The ECL system is a chemiluminescence-based detection system used in western blotting applications to visualize and quantify proteins. The system generates a luminescent signal proportional to the amount of target protein present on the membrane, allowing for sensitive and accurate protein detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (121 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (29 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (27 mentions)
Ripa lysis buffer, by Beyotime (24 mentions)
Bca protein assay kit, by Beyotime (20 mentions)
Ripa buffer, by Thermo Fisher Scientific (17 mentions)
Gapdh, by Cell Signaling Technology (14 mentions)
Pvdf membrane, by Bio-Rad (14 mentions)
Gapdh, by Santa Cruz Biotechnology (13 mentions)
Bca kit, by Beyotime (12 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (12 mentions)
Bca kit, by Thermo Fisher Scientific (12 mentions)
Ripa buffer, by Merck Group (11 mentions)
Ripa buffer, by Beyotime (11 mentions)
Transfer apparatus, by Bio-Rad (9 mentions)
Image lab software, by Bio-Rad (9 mentions)
β actin, by Cell Signaling Technology (9 mentions)
β actin, by Merck Group (9 mentions)
Protease inhibitor, by Roche (9 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (9 mentions)
448 protocols using ecl system
1
Cas9 Protein Expression Analysis
2
Western Blot Analysis of EMT Markers
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Cells were lysed in lysis buffer and proteins (30 µg) were separated on 10–12% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking membranes, they were incubated with appropriate dilutions (1:2000 for internal control antibodies and 1:1000 for others) of specific primary antibodies against β-tubulin, GAPDH, β-Catenin, ZEB1, ZO-1, N-cadherin, E-cadherin, Vimentin, Slug, Snail, Claudin-1 (from CST). The blots were incubated with HRP-conjugated secondary antibodies and visualized using the ECL system (Thermo Fisher Scientific, Rochester, NY).
3
Protein Expression Analysis via Western Blot
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We follow the method with reported work[15 (link)]. Cells were lysed in RIPA buffer, and an equivalent of 40 µg total protein was separated on 10% SDS-PAGE and transferred onto 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked and probed with specific primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. The immunoreactive bands were visualized using the ECL system (Thermo Fisher Scientific).
4
Western Blot Protein Detection
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According to a previous study [28 (link)], protein extracts were loaded onto 10% SDS gel and electrophoresed. The protein extracts were then transferred to the PVDF membrane (Millipore) and incubated with primary antibodies at 4°C overnight. The next day, the membrane was incubated with secondary antibodies for 2 h at room temperature. Finally, the protein bands were captured using ECL system (Thermo Fisher Scientific, Inc.).
5
Protein Expression Analysis in Brain Regions
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Forebrain, nucleus accumbens, and dorsal striatum tissue samples were homogenized in RIPA buffer (Cell Signaling Technology, Danvers, MA) containing a cocktail of protease and phosphatase inhibitors (MilliporeSigma). The concentration of total protein was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Equal amounts of total protein were separated electrophoretically on an SDS-PAGE gel, transferred to a PVDF membrane (Bio-Rad, Hercules, CA) and immunoblotted with antibodies: anti-DAT (MilliporeSigma), anti-D2R (MilliporeSigma), anti-TH (MilliporeSigma), anti-actin (MilliporeSigma), anti-GAPDH (MilliporeSigma), and anti-TN. Blots were developed with the ECL system (Thermo Fisher Scientific). Band intensities were quantified from digital images by densitometry using Image J.
6
Western Blot Analysis of HURP Protein
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Briefly, the NSCLC cell lines were lysed with RIPA buffer containing protease inhibitor. Following determination of the protein concentrations using the BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China), equal amounts of protein per sample (20 µg) were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels for 2 h and transferred to nitrocellulose membranes. The membranes were subsequent blocked with 5% low-fat milk in TBST for 1 h and incubated with primary antibodies overnight at 4°C. The primary antibodies included: anti-HURP (1:2,000; Abcam) and GAPDH (1:2,000; Santa Cruz Biotechnology). This was followed by incubation with secondary antibodies (1:5,000; Santa Cruz Biotechnology) for 1.5 h at room temperature and rinsed four times with TBST for 8 min each. Reactions were visualized using the ECL system (Thermo Fisher Scientific, Waltham, MA, USA).
7
Western Blot Analysis of Viral Proteins
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At post-infection day 28, cells were lysed in SDS-Lysis buffer (1M Tris-HCl pH 6.8, 2% SDS, 30% Glycerol) supplemented with protease inhibitors (Roche, #04693132001). The concentrations of whole cell lysates were measured using the Pierce BCA protein assay kit (Thermo Scientific, #23227). Equal amounts of whole cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, #10600006) using a transfer apparatus according to the manufacturer’s protocols (Bio-rad). After incubation with 5% BSA in TBS containing 0.1% Tween-20 (TBST) for 30 min, the membrane was incubated with primary antibodies at 4°C overnight; MW8 (Developmental Studies Hybridoma Bank, 1:500) and MW1 (Developmental Studies Hybridoma Bank, 1:500). Following incubation, membranes were incubated with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody for 1 hr. Blots were developed with the ECL system (Thermo Scientific, #34080) according to the manufacturer’s protocols.
8
Quantifying pCREB Levels in Response to E2 and BPA
9
Western Blot Analysis of Protein Lysates
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Cells were lysed with Pro-Prep protein extraction solution (Intron Biotechnology) on ice for 1 h. After centrifugation at 16,000 × g for 30 min, the supernatant was harvested. The concentration of protein lysates was determined by Bradford protein assay (Bio-Rad). Proteins were loaded onto an SDS-PAGE gel (Elpis Biotech, Daejeon, Korea) and then transferred to a nitrocellulose membrane (Whatman, Maidstone, England). The membrane was blocked with TBST (0.1 % Tween in TBS) containing 4 % skim milk, washed in TBST, and treated with the appropriate primary antibodies. The primary antibodies used in this study were as follows: SMAD2 (Cell Signaling); p-SMAD2 (Cell Signaling); ACTIN (Santa Cruz Biotechnology); ATP7A (Hycult Biotech, Uden, Netherlands); SMAD1 (Cell Signaling); and p-SMAD1 (Cell Signaling). After washing in TBST, the membrane was incubated with an horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. The membrane was developed using the ECL system (Thermo Fischer Scientific), and images were captured by LAS-3000 (Fuji Film, Tokyo, Japan).
10
Recombinant YB-1 Protein Serum Binding
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Recombinant YB-1 proteins were incubated with 0.1 µl human serum for 30′, 1 h, and 16 h at 37 °C. Afterward separated by SDS-electrophoresis, blotted on nitrocellulose membrane, and incubated overnight at 4 °C with primary polyclonal anti-YB-1 antibody (Sigma-Aldrich; 1:1000) or monoclonal anti-Flag antibody (Flag M2, Sigma-Aldrich; 1:2000). Peroxidase-conjugated secondary goat-anti-rabbit-antibody (Southern Biotech; 1:5000) and the ECL system (Thermo Fisher Scientific) were used for detection.
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