Binding of mAbs to RV protein was analyzed on SDS-PAGE (4–20% polyacrylamide; Mini Protean TGX gels; Bio-Rad) under reducing conditions. Briefly, the protein samples were boiled for 5 minutes at 100 °C in 2× Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol and subjected to SDS-PAGE electrophoresis, and then transferred onto nitrocellulose membranes using the iBlot 2 dry blot system (Thermo Fisher) at 20 V for 7 min. Following blocking with 3% skim milk for 2 h at RT, the membrane was incubated with primary antibody overnight at 4 °C. After three washes with 1×PBST buffer (1× phosphate-buffered saline, 0.05% Tween 20), HRP-conjugated goat anti-mouse IgG (abcam) was added to the membrane and incubated at RT for 1 h. The positive bands were visualized by electrochemiluminescence (ECL) reagent (Life Technologies) and developed using1-Step Ultra TMB-Blotting Solution (Life Technologies).
Ecl reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Japan, Germany, France
ECL reagent is a chemiluminescent detection solution used for Western blot analysis. It is designed to enable sensitive detection of target proteins on membranes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (123 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (102 mentions)
Bca kit, by Thermo Fisher Scientific (43 mentions)
Ripa lysis buffer, by Beyotime (37 mentions)
Bca protein assay kit, by Beyotime (34 mentions)
Ripa buffer, by Thermo Fisher Scientific (29 mentions)
Ripa buffer, by Beyotime (28 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (27 mentions)
Bca protein assay, by Thermo Fisher Scientific (27 mentions)
Pvdf membrane, by Bio-Rad (23 mentions)
Polyvinylidene difluoride membrane, by Merck Group (23 mentions)
β actin, by Santa Cruz Biotechnology (17 mentions)
β actin, by Cell Signaling Technology (17 mentions)
β actin, by Merck Group (17 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (17 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (16 mentions)
Protease inhibitor cocktail, by Roche (16 mentions)
Quantity one software, by Bio-Rad (15 mentions)
Gapdh, by Abcam (15 mentions)
β actin, by Abcam (15 mentions)
921 protocols using ecl reagent
1
SDS-PAGE Analysis of mAb Binding to RV Protein
2
Western Blot Analysis of Myc, MLKL, and GAPDH Proteins
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Whole cell extracts were lysed on ice in RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 7.4, 1% NP-40, 0.1% SDS), supplemented with complete EDTA-free protease inhibitor cocktail (Roche). Total protein concentrations were quantified using the BCA protein kit (Life Technologies), and cell lysates containing 20 μg of protein were subjected to electrophoretic separation on denaturing polyacrylamide gels under reducing conditions, followed by transfer to PVDF membranes. The latter were then probed with a mouse anti-myc antibody (1:1000, Cell Signalling Technologies), a rat anti-MLKL antibody (1:2000) [15 (link)] and a rabbit anti-GAPDH antibody (1:2500, Trevigen), followed by the appropriate secondary horseradish peroxidase-conjugated antibody (1:3000, Cell Signalling Technologies). The signal was visualized using the chemiluminescent ECL reagent (Life Technologies).
3
Western Blot Protein Detection
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Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane using a semidry Western blotting apparatus (Bio-Rad). The membranes were blocked with PBS containing 5% nonfat milk for 1 h at ambient temperature. After blocking, the membranes were probed with primary antibodies overnight at 4°C. The membranes were washed four times with blocking buffer before adding HRP-conjugated secondary antibodies (Life Technologies), diluting as recommended in blocking buffer, and incubating for 30 min at ambient temperature. After washing three times with PBS containing 0.1% Tween 20 (Sigma-Aldrich), the membranes were developed using ECL reagent (Life Technologies) and imaged on the ChemiDoc system (Bio-Rad) or by exposing to scientific imaging film (Kodak).
4
Western Blot Analysis of Cellular Proteins
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Western blotting was performed as described previously [19] . Brie y, Cells were collected and washed three times with a phosphate buffer (PBS). Extraction of cells were lysated by Ripa buffer containing protease and phosphatase inhibitor cocktail (Sigma, St Louis, MO). The homogenate was centrifuged at 4 ℃ at 10000 × g for 20 minutes. The supernatant was collected and the protein concentration was determined by Bradford protein test kit (Bio-Rad, Hercules, USA). Samples were separated on 8-12 % SDS-PAGE gels, and then transferred onto PVDF membrane. According to the primary antibody, the imprinted membrane was sealed with 5 % milk or 5 % bovine serum albumin for 30 minutes, followed by incubation with primary antibodies against Caspase-3, E-cadherin, ZO-1, Claudin-5 or N-cadherin etc. (1: 1000 dilution), and β-actin (1: 5000 dilution), respectively. After that, the membrane was incubated horse radish peroxidase (HRP)-conjugated secondary antibodies (1: 5000, Abcam, Cambridge, MA). After developing the membrane with ECL reagent (Life Technologies, Grand Island, NY), the expression of the protein was observed.
5
Western Blot Protocol for BLM Protein
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Cells were lysed with RIPA buffer (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 0.25% sodium deoxycholate) and quantified using the BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts (40 µg) protein were separated on a 10% SDS-polyacrylamide gel and electrotransferred to PVDF membranes (MilliporeSigma). Membranes were immersed in blocking buffer with 10% fat free milk for 1 h at room temperature. The blocked PVDF membrane was incubated with antibodies against BLM (1:1,000; cat. no. 2742; Cell Signaling Technology, Inc.) for 3 h at room temperature. Following incubation with the primary antibodies, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies for 1 h (1:5,000; cat. nos. 31466 and PA174421; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature. Protein bands were detected by using enhanced chemiluminescence. β-actin (1:1,000; cat. no. 3700; Cell Signaling Technology, Inc.) was used as the loading control. Proteins bands were visualized using an ECL reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the immunoblots were quantified using ImageJ software (version 3.0; National Institutes of Health).
6
Survivin Protein Immunoblot Assay
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Purified recombinant human survivin (Abcam #ab87202) or survivin peptide (aa 53-67 or aa53-67/M57) were bound to nitrocellulose membrane using the Minifold II slot-blot system (Schleicher & Schuell). Membranes were dried, blocked in 5% milk in TBST for 30 minutes, then incubated overnight with anti-survivin antibody 60.11 (Novus, Littleton, CO) diluted 1:500 in blocking buffer. Membranes were washed in TBST, incubated with HRP-conjugated secondary anti-mouse antibody, washed again, incubated with ECL reagent (Invitrogen), then exposed to X-ray film.
7
Investigating CyPA-induced Myocardial Protein Levels
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The ability of SP‐8356 to inhibit CyPA‐induced expression and corresponding protein levels of MMP‐9, ERK1/2, p‐ERK1/2, P65 and p‐P65 in primary rat myocardiocytes was assessed using Western blotting. The samples were added to the RIPA buffer, homogenized and centrifuged. The supernatant layer was collected. Protein levels were measured by the bicinchoninic acid kit (Thermo Fisher Scientific). The proteins extracted from the primary rat myocardiocytes were electrophoresed in SDS‐PAGE using the Invitrogen apparatus (Thermo Fisher Scientific) at a constant voltage. In the next step, the separated proteins were blotted to a PVDF membrane (EMD Millipore) and blocked using 5% skim milk, and the blot was incubated with specific primary antibodies against MMP‐9, ERK1/2, p‐ERK1/2, P65 and p‐P65. As a secondary antibody, a Horseradish peroxidase goat anti‐mouse immunoglobulin (BD Bioscience) was used. The protein bands were developed using the enhanced chemiluminescence (ECL) reagent (Invitrogen).
8
Western Blot Analysis of SNAI1, IRS, and HMGA2
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The collected samples were first lysed in a RIPA buffer (Invitrogen, Carlsbad, CA) in strict accordance with the standard protocol recommended by the manufacturer on the product manual. Then, the sample proteins were resolved by 10% SDS-PAGE and blotted onto a NC membrane, which was blocked with 5% skim milk and incubated sequentially with primary anti-SNAI1, anti-IRS, and anti-HMGA2 antibodies as well as HRP-tagged secondary antibodies (Abcam, Cambridge, MA) in strict accordance with the incubation conditions recommended by the antibody manufacturer on the product manual. Finally, the color of protein bands was developed by making use of an ECL reagent (Invitrogen, Carlsbad, CA) in strict accordance with the standard protocol recommended by the manufacturer on the product manual and the relative expression of SNAI1, IRS, and HMGA2 proteins in each sample was calculated by using the Volume One software (Bio-Rad laboratories, Hercules, CA).
9
Evaluation of Inflammatory Biomarkers in Carbamazepine Therapy
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The following materials and instruments were purchased from commercial vendors: carbamazepine tablets from Novartis (CFDA approval no. H11022279; Shanghai, China); vitamin B12 tablets from Guangdong Yixiang Pharmaceuticals (CFDA approval no. H44020621; Guangdong, China); TNF-α ELISA kit, hs-CRP ELISA kit, TRIzol kit, reverse transcription kit, and ECL reagent from Invitrogen (Carlsbad, CA, USA); human homocysteine assay kit from B&D Systems (Bedford, MA, USA); primary rabbit anti-human TNF-α, hs-CRP, and gAPDH polyclonal antibodies (1:1,000; cat. nos. 6945, 14316 and 2118), and horseradish peroxidase conjugated mouse anti-rabbit secondary polyclonalantibody (1:2,000; cat. no. 93702) from Cell Signaling Technology (Beverly, MA, USA); PCR instrument from Applied Biosystems (Foster, CA, USA); UV imaging system and electronic balance (BP121S) from Biometra (Goettingen, Germany). Other instruments and reagents are described in relevant context.
10
Radiochemical Synthesis and Characterization
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Propidium iodide, Dulbecco’s Modified Eagle’s Medium (DMEM), RNAse, secondary Goat anti-Rabbit antibody, ECL reagent and Diaminobenzidine (DAB) were procured from Invitrogen, Carlsbad, CA, USA. Glutathione, Sulphuric acid, Diethylenetriaminpentaacetic acid (DTPA) anhydride, stannous chloride, sodium bicarbonate, 3, (4, 5-dimethylthiazol-2-yl)2, 5-diphenyltetrazolium bromide (MTT), Sulforhodamine B (SRB), Glacial acetic acid, Trichloroacetic acid (TCA), pyridine, Harri’s Hematoxylin, acetone, Xylene, DPX, sodium citrate, Trypsin-EDTA, Bovine serum albumin (BSA), Hank’s balanced salt solution (HBSS), Total Glutathione estimation kit were purchased from Sigma Aldrich, St Louis, MO, USA. Primary anti GGT polyclonal rabbit antibody was purchased from Abcam. Ethanol, methanol, dimethyl sulfoxide, dimethyl formamide, diethyl ether, triethylamaine were obtained from Merck specialities pvt. limited. All solvents used were of analytical grades. Radiocomplexation and the radiochemical purity were checked by instant thin layer chromatography (ITLC-SG) Strips from Paul Gelman, USA.
99mTc was procured from Regional Center for Radiopharmaceuticals (Northern Region), Board of Radiation and Isotope Technology (BRIT), Department of Atomic Energy, India. 68Ga was eluted from in house 68Ge/68 Ga generator system (IGG100, Eckert and Ziegler, Berlin).
99mTc was procured from Regional Center for Radiopharmaceuticals (Northern Region), Board of Radiation and Isotope Technology (BRIT), Department of Atomic Energy, India. 68Ga was eluted from in house 68Ge/68 Ga generator system (IGG100, Eckert and Ziegler, Berlin).
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