Total protein of tissues and cells was obtained using radioimmunoprecipitation assay lysis buffer containing a mixture of proteinase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was determined using BCA protein assay reagent (Beyotime Institute of Biotechnology, Haimen, China). Equivalent amounts of proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membranes (Invitrogen; Thermo Fisher Scientific, Inc.). After being blocked in Tris-buffered saline containing 5% fat-free milk, the membranes were incubated with the aforementioned VCAM-1, ICAM-1, IL-6, IL-8, NF-κB p65, ERK or p-ERK primary antibodies at 4°C overnight, washed using phosphate-buffered saline (PBS)-Tween buffer [0.01 mol/l PBS (pH 7.2–7.4); 0.2 mol/l NaH2PO4 19 ml; 0.2 mol/l Na2HPO4 81 ml; NaCl 17 g; 2,000 ml ddH2O; 20% Tween 20] and then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. A β-actin antibody was used as a loading control. Signals were detected on X-ray film using the Pierce ECL detection system (Thermo Fisher Scientific, Inc.).
Pierce ecl detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce ECL detection system is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It generates a luminescent signal when exposed to horseradish peroxidase (HRP)-labeled antibodies, allowing for the visualization of target proteins on a photographic film or digital imager.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Image lab, by Bio-Rad (3 mentions)
Tris glycine gel, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (2 mentions)
Iblot, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (2 mentions)
Power blotter select transfer stacks, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor, by Merck Group (1 mentions)
Bca protein assay reagent, by Beyotime (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Phospho foxo1, by Cell Signaling Technology (1 mentions)
Actin antibodies, by Merck Group (1 mentions)
13 protocols using pierce ecl detection system
1
Protein Expression Analysis of Inflammatory Markers
2
Immunoblotting Analysis of TcpA Expression
3
Protein Expression Analysis of PDLCs
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Whole-cell lysates of PDLCs were prepared with a RIPA buffer (Thermo) containing a protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was quantised using the BCA protein assay (Beyotime). Total protein was evaluated by WB with primary antibodies against RUNX2 (#sc-390715, 200 µg/mL, SANTA), alkaline phosphatase (ALP; #sc-365765, 1:1000, SANTA), OSX (#sc-393325, 1:1000, SANTA), SPRY1 (#sc-365520, 200 µg/mL, SANTA), and β-actin (#sc-81178, 1:200, SANTA) as previously described.20 Protein bands were visualised with Pierce ECL detection system (Thermo), followed by analysing the intensity using Image Lab (Bio-Rad).
4
Immunoblotting Analysis of FOXO1 and AKT
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Cells were incubated for 30 min in NP-40 lysis buffer [20 mM Tris pH 7.5 containing 140 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P-40, 5 μM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1.5 nM aprotinin, 10 nM E-64, and 10 nM leupeptin]. The cells were then sonicated and centrifuged at 12,000 × g for 10 min at 4°C to remove insoluble debris. Total proteins (30 μg) were resolved in an SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane using the semidry technique. The membranes were blotted with antibodies against FOXO1, phospho-FOXO1, AKT, and phospho-AKT (Cell Signaling Technology, USA), and the proteins were identified using the Pierce ECL detection system (Thermo Scientific, USA).
5
BRCA1 and ALDH1 Protein Detection
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Total cell lysates were prepared by lysing the cells in modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP-40) supplemented with 100 μg/ml leupeptin, 100 μg/ml aprotinin and 1 mM sodium orthovanadate. BRCA1 and ALDH1 antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA., Actin antibodies were from Sigma (St. Louis MO) and EpCAM antibodies were from AbCam. HRP conjugated Trueblot secondary antibodies were purchased from eBioscience (eBioscience Inc. San Diego, CA) and western blots were developed using a Pierce ECL detection system (Thermo Scientific, Rockford IL).
6
Western Blot Analysis of Signaling Proteins
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After incubation cells or tissue were lysed in sample buffer containing 50 mmol/L Tris-HCl pH 6.8, 2% w/v sodium dodecyl sulfate, 10% glycerol, 0.0025% w/v bromophenol blue, and 5% β-mercaptoethanol. Protein samples were then loaded and resolved on Mini-PROTEAN TGX Gels and then transferred onto polyvinylidene fluoride membranes (BioRad) using a Trans-Blot Turbo Transfer System (BioRad). The polyvinylidene fluoride membranes were then blocked with 10% nonfat dry milk or BSA in PBS with 0.1% v/v Tween (PBS-T) for 1 hour, and then incubated with the primary antibodies. Membranes were developed with the Pierce ECL detection system (Thermo-Fisher Scientific) and signal detected using photographic film (GE Healthcare, catalog no. 28-9068-37) and an automatic processor (Fuji RG II). Samples were immunoblotted for Phospho-VASP (Ser239; Cell Signaling, No. 3114), Phospho-VASP (Ser157; Cell Signaling, no. 84519), cGAS (Invitrogen, no. PA5-76367), Phospho-TBK1 (Ser172; Cell Signaling, no. 5483), TBK1 (Cell Signaling, no. 3013), Phospho-STING (Ser365; Cell Signaling, no. 72971), CD31 (Abcam, no. ab28364), MRP1 (Santa Cruz, no. sc-18835), LRRC8A (Santa Cruz, no. sc-517113), GAPDH (Cell Signaling, no. 2118), and β-actin (Sigma-Aldrich, no. A5316).
7
Immunoprecipitation and Western Blot Analysis of Hoxa3 Protein
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Lysates from mφs treated with Hoxa3mCh or mCherry CM were prepared using IP lysis buffer (50 mM Tris-acetate [pH 7.5], 300 mM NaCl, 1 mg/ml BSA, 2% Igepal, 1 mM EDTA, 1× protease inhibitor mixture [Promega], and 1× sodium orthovanadate). Two micrograms of rabbit anti-mCherry (BioVision) was labeled with biotin (DSB-X; Molecular Probes) and incubated with 50 μg protein lysate, together with 0.4 mg prewashed Dynabeads (FlowComp Flexi system; Invitrogen). The mixture was left overnight at 4°C on a rotator before washing and elution with immunoprecipitation wash buffer (0.1% SDS, 500 mM Tris-acetate [pH 7.5], 300 mM NaCl, and 0.5% Igepal) using a magnet (EasySep; StemCell Technologies).
CM from 293T cells transfected with SP-Hoxa3mCh or SP-mCherry and purified lysate from mφs treated with CM were analyzed by Western blotting using 10% SDS-PAGE and polyvinylidene difluoride membrane, using the Bio-Rad Turbo Transfer System. Blots were probed at 4°C overnight with 0.1 μg rabbit anti-mCherry Ab in a volume of 6 ml and visualized using the Pierce ECL detection system (Thermo Fisher Scientific). Images were generated using the ChemiDoc MP System (Bio-Rad) and Image Lab Software.
CM from 293T cells transfected with SP-Hoxa3mCh or SP-mCherry and purified lysate from mφs treated with CM were analyzed by Western blotting using 10% SDS-PAGE and polyvinylidene difluoride membrane, using the Bio-Rad Turbo Transfer System. Blots were probed at 4°C overnight with 0.1 μg rabbit anti-mCherry Ab in a volume of 6 ml and visualized using the Pierce ECL detection system (Thermo Fisher Scientific). Images were generated using the ChemiDoc MP System (Bio-Rad) and Image Lab Software.
8
Signaling Assays and Co-Immunoprecipitation Protocol
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For signaling assays, NCI-H1299 and NCI-H1792 cells were seeded at confluence and serum starved overnight. Cells were lysed in RIPA buffer (Sigma) and the lysates Western blotted on a 4-15% Tris-glycine gel and transferred to a 0.2μm nitrocellulose using a Mini-PROTEAN electrophoresis system (Bio-Rad Laboratories, Hercules, CA). For co-immunoprecipitation, HEK 293T cells were transfected as above and lysed in modified RIPA buffer (150mM NaCl, 50mM Tris, pH 7.5, 1% NP-40). Precleared lysates were immunoprecipitated with GFP-Trap agarose beads (Allele Biotech, San Diego CA) or primary antibody as appropriate and washed with lysis buffer. Western blots were developed using a Pierce ECL detection system (Thermo Scientific, Rockford IL) and autoradiography film (MidSci, St. Louis, MO).
9
Quantification of Apoptosis-Related Proteins
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Cells were incubated for 30 min in NP-40 lysis buffer [20 mM Tris pH 7.5 containing 140 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P-40, 5 μM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 1.5 nM aprotinin, 10 nM E-64, and 10 nM leupeptin]. The cells were then sonicated and centrifuged at 12,000 × g for 10 min at 4°C to remove insoluble debris. Protein concentration was determined by the Bradford method. Total proteins (30 μg) were resolved on 10% to 12% SDS-PAGE gels. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA), immunoblotted with primary antibodies [Bax, Bcl-xL, LC3II, and β-actin (Cell Signaling Technology, USA)], and detected with peroxidase-linked antibodies and a Pierce ECL detection system (Thermo Scientific, USA).
10
Protein Extraction and Western Blot Analysis
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Cells were lysed with a cell lysis buffer (60 mM 1-O-n-octyl-β-D-glucopyranoside, 0.15 M NaCl, 10 mM HEPES-NaOH, pH 7.2, 1/100× protease inhibitor cocktail [Nacalai, Kyoto, Japan]), and centrifuged at 20,000 g for 30 minutes. The supernatant was collected, mixed with sodium dodecyl sulfate (SDS) gel sample buffer supplemented with or without 10% β-mercaptoethanol, and loaded onto 15% SDS electrophoresis gels. Separated proteins were electrotransferred to a polyvinylidene difluoride membrane (Immobilon-P; Merck Millipore, Darmstadt, Germany), and the membrane was blocked in Tween 20-Tris Buffered Saline (TTBS) (10 mM Tris-HCl pH 7.5, 0.15 M NaCl, 0.05% Tween20) containing 1% skim milk for 1 hour, followed by incubation with a primary antibody (at 1 μg/mL if not indicated) in TTBS for 1 hour. The membrane was then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000) for 1 hour. Chemiluminescent detection was carried out with the Pierce ECL detection system (Thermo Fisher Scientific) according to the manufacturer's instructions.
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