Western blot analysis were done with the following antibodies: p-CHK1 S345 (#2348, 1:2,000, Cell Signaling), total CHK1 (#04-207, 1:1,000, Millipore), p-CHK2 T68 (#2197, 1:2,000, Cell Signaling), total CHK2 (#6334, 1:1,000, Cell Signaling), p-ATM S1981 (#5883, 1:1,000, Cell Signaling), total ATM (#2873, 1:1,000, Cell Signaling), p-ATR S428 (#2853, 1:1,000, Cell Signaling), total ATR (#2790, 1:1,000, Cell Signaling), p-p53 S15 (#9286, 1:2,000, Cell Signaling), total p53 (#OP03, AB-1, 1:5,000, Oncogene), cleaved Caspase3 (#9661, 1:2,000, Cell Signaling), PARP (#9542, 1:10,000, Cell Signaling), β-actin (#A5316, 1:10,000, Sigma Aldrich), Vinculin (3AB73412, 1:5,000, Abcam). Protein bands were visualized using chemiluminescence substrates (#170-5061, BioRad) and imaged on Kodak X-OMAT 2000A.
Chemiluminescence substrate
Manufactured by Bio-Rad
Sourced in United States
Chemiluminescence substrate is a reagent used in Western blotting and other immunoassay techniques to detect and quantify proteins. It generates a luminescent signal when it reacts with the enzyme-labeled detection antibody, allowing for the visualization and analysis of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
L8 ultracentrifuge, by Beckman Coulter (2 mentions)
Chemidoc xrs imager, by Bio-Rad (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Versadoc imaging system, by Bio-Rad (2 mentions)
Non fat dry milk, by Merck Group (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Chemostar imaging system, by Intas (2 mentions)
Mouse anti ipad monoclonal antibody, by Abbexa (2 mentions)
Anti mouse secondary antibody conjugated with hrp igg hrp, by Thermo Fisher Scientific (2 mentions)
Semi dry transfer apparatus, by Bio-Rad (2 mentions)
Tween 20, by Merck Group (2 mentions)
28 protocols using chemiluminescence substrate
1
Comprehensive Western Blot Analysis
2
Protein Expression Analysis in HUVECs
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Cultured HUVECs were collected in RIPA buffer to isolate total proteins (110 ). Equal amount of proteins from each sample were loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels, which were then subjected to electrophoresis. Proteins were then transferred onto PVDF membranes (Bio-Rad), and the following antibodies were employed to detect for the proteins of interest [Cell Signaling Technology: ICAM-1 (4915S, dilution 1:1000), VCAM-1 (13662S, dilution 1:1000), eNOS (32027S, dilution 1:1000), phospho (p)-eNOS (Millipore, 07 -428-I, dilution 1:1000), AKT (4691S, dilution 1:1000), p-AKT (4060S, dilution 1:1000), cleaved-CASPASE3 (9664S, dilution 1:1000), p21 (2947S, dilution 1:1000), and GAPDH (5174S, dilution 1:1000)]. Western blot for FABP3 was performed using polyclonal antibody (ThermoFisher, PA5-92386, dilution 1:1000), and wildtype mouse total heart protein was used as a positive control. Western blots were developed using chemiluminescence substrates (Bio-Rad) and the Licor-Odyssey XF Imaging System. Densitometry was performed to measure the band intensities using the Image Studio Lite.
3
Western Blot Analysis of UCP2 and Ubiquitin
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The cells subjected to the indicated treatments were lysed by the Laemmli sample buffer as previously described (16 (link),19 (link)). The protein of lysate was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis was performed as previously described (19 (link)). Briefly, following SDS-PAGE, the separated proteins were transferred onto PVDF membranes and probed with rabbit anti-UCP2 antibody and mouse anti-ubiquitin (sc-166553) (both from Santa Cruz Biotechnology, Inc.). Specific reactions were detected using goat anti-rabbit IgG or goat anti-mouse IgG conjugated with horseradish peroxidase (Sigma, St. Louis, MO, USA) and revealed by a chemiluminescence substrate (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were also blotted with tubulin antibody (sc-33749; Santa Cruz Biotechnology, Inc.) to normalize protein loading. The chemiluminescence signal was recorded using the ChemiDoc MP imaging system (Bio-Rad Laboratories). The luminescence signal was captured and analyzed using the Image Lab Program (version 6.1).
4
Quantification of Protein Expression
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Following 72 h of treatment, cells were lysed and the extracted proteins were quantified using DC protein assay kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Proteins were resolved on 8% or 12% SDS-poly-acrylamide gels, then transferred onto a nitrocellulose membrane using a semi-dry transfer cell. The membranes were blocked in TBS-T containing 5% non-fat milk for 1 h and incubated overnight at 4oC overnight with indicated primary antibodies (Cell Signaling Technologies, Beverly, MA, USA) and then incubated with goat anti-mouse or anti-rabbit secondary antibody (Cell Signaling Technologies, Beverly, MA, USA) for 1 h at room temperature. Detection was done using chemiluminescence substrate (Bio-Rad, Hercules, CA, USA). β-actin (Cell Signaling Technologies, Beverly, MA, USA) was used as a loading control.
The blots were captured with ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) and bands were visualized with the Image Lab v5.2.1 software (Bio-Rad, Hercules, CA, USA).
The blots were captured with ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) and bands were visualized with the Image Lab v5.2.1 software (Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis of Retinal Proteins
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The protein expression of Sirt1, acetyl-p53, BDNF, and TrkB in the whole retina were analyzed by western blot. After mice eyeballs were enucleated, the retina and sclera were isolated. Protein lysates were extracted using RIPA Lysis Buffer (Thermo Fisher Scientific) supplemented with protease inhibitor (Sigma-Aldrich), according to the manufacturers’ protocol. Briefly, all lysates were quantified, and equal volumes were loaded on NuPAGE 4–12% Bis-Tris gel (Invitrogen) before being transferred to polyvinylidene difluoride membranes (Invitrogen). Membranes were blocked with 3% skim milk and incubated with anti-Sirt1 primary antibodies (1:1000; Cell Signaling Technology), anti-acetyl-p53 (1:400; Abcam), rabbit anti-BDNF antibody (1:500; GeneTex, San Antonio, TX, USA), and rabbit anti-TrkB antibody (1:1000; Abcam) overnight at 4°C. Bound proteins were detected using peroxidase-conjugated ECL anti-mouse IgG and anti-rabbit (GE Healthcare UK). Proteins were visualized using a chemiluminescence substrate (Bio-Rad Laboratories) and imaging system (DNR Bio imaging systems, Neve Yamin, Israel). Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The expression ratios of protein bands were quantified using ImageJ, as previous described.68
6
Evaluating Cellular Signaling Pathways
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The HUVECs were lysed with the RIPA solution (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0), to which we added a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). In each sample, the protein concentration was evaluated using a Bradford assay. Proteins (25 µg) were separated by 4–15% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA), and then transferred to a 0.2 mm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane, which was then incubated overnight with primary antibodies. Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-ICAM-1 (Cell Signaling), mouse anti-β-actin, mouse anti-α-tubulin, and rabbit-anti-GAPDH (Cell Signaling) were used as the primary antibodies. Anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies were used as the secondary antibodies (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to distinguish the protein bands, using a chemiluminescence substrate (Bio-Rad), that were then quantified using ImageJ software. Each measure was normalized versus β-actin, α-tubulin, or GAPDH.
7
Western Blot Analysis of Protein Aggregates
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Five microliters of lysate and 25 µL of bead eluent were resolved on 4 to 20% gradient Bis-Tris gels (ThermoFisher) and blotted onto activated polyvinylidene difluoride using wet transfer with settings at 120 V for 1 h. Following transfer, membranes were rinsed with PBS and then fixed with 4% paraformaldehyde as previously described (41 (link)). Membranes were then dried completely and reactivated with methanol just prior to use. Membranes were blocked in TBST containing 5% bovine serum albumin for 2 h at room temperature. Membranes were then incubated overnight at 4 °C with blocking buffer containing one of the following antibodies: 1) anti–alpha-synuclein “SYN1” (BD Lifesciences) and 2) “MJFR1” (Abcam), 3) anti-ubiquitin conjugates “UBCJ2” (Enzo Life Sciences), and 4) anti-PSER129 “EP1536Y” (Abcam). To determine enrichment of biotinylated proteins, one membrane was incubated with prepared ABC reagent, diluted 1:10 in blocking buffer for 1 h at room temperature (Vector Laboratories). The next day, membranes were washed three times for 10 min each time with TBST and incubated for 1 h with either anti-rabbit (1:5,000) or anti-mouse (1:10,000) HRP-conjugated secondary antibodies (Cell Signaling Technologies) diluted in blocking buffer. Membranes were then washed three times for 10 min each time with TBST and detected using chemiluminescence substrate (Bio-Rad).
8
Western Blot Protein Detection
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Cell lysates were extracted with the lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1 mM DTT, 0.2 mM PMSF, 1 ug/ml aproptinin, 1 ug/ml leupeptin, 1 mM Na3VO4 and 1 mM NaF), separated on 10 % SDS-PAGE gel, and transferred onto the nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was blocked with 5 % BSA in TBST (20 mM Tris-HCl pH 7.6, 137 mM NaCl and 0.1 % Tween-20), probed with specific primary antibody at 4 °C overnight, and bound with HRP-conjugated secondary antibody (Bio-Rad) at room temperature for 1 h. The membrane was developed with a chemi-luminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA), and the blot was autographed onto a X-ray film (Fuji Photo Film Co., Japan).
9
Prepore Production and Characterization
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Prepore was produced by two separate methods that were published [1 (link)] [2 (link)]. Briefly, in the first method, 1 µg protoxin was mixed with 10 µg insect brush border membrane vesicles (BBMV) in the presence of 50 µl of solubilization buffer (50 mM Na2CO3 pH 10.5 + 0.2% β-mercaptoethanol) and incubated at room temperature (25°C – 28°C) for 15 min. In the second method, 200 ng of protoxin was incubated with scFv73 in a 1:2 mass ratio and 5% midgut juice was added in 100 µl of solubilization buffer containing 60 µM small unilamelar vesicles made of 1,2-Dioleoyl-sn-Glycero-3-phosphocholine (DOPC). The mixture was incubated at 37°C for 1 hr, followed by precipitating the membrane bound toxin at 400,000× g in a Beckman L8 ultracentrifuge. A control reaction, which lacked any small unilamellar vesicles, was used to show no precipitate formation in the absence of SUV. The pellet was resuspended in 50 µl of buffer in presence of 10% n-octyl-β-D-glucopyranoside and clarified by centrifugation, treated with loading dye and boiled for 3 – 5 min. Western blot analysis of the protein was performed using polyclonal anti-prepore antibody (1:50,000; 1hr) and secondary HRP antibody (1:10,000; 1hr) and was detected using chemiluminescence substrate (Bio-Rad).
10
Western Blot Analysis of Cellular Proteins
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Cell lysates were separated on 10% SDS-PAGE. The nitrocellulose membrane was blocked with 5% skim milk in TBST (20 mM Tris-HCl pH 7.6, 137 mM NaCl and 0.1% Tween-20), probed with the indicated primary antibody, such as anti-INSM1, anti-N-myc (Santa Cruz Biotech.), anti-pAKT, AKT (S-473), pGSK3β (S-9) and GSK3β (Cell Signaling Tech.), anti-phospho-Myc (pT-58) (Abgene) and anti-GAPDH at 4°C overnight and bound with HRP-conjugated secondary antibody (Bio-Rad) at room temperature for 1 h. A chemiluminescence substrate (Bio-Rad) was autographed onto a X-ray film.
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