Whole astrocyte lysates were prepared by incubating cells on ice for 30 min in buffer containing 100 mM PBS, 2 mM NaF, 2.5 mM Na4P2O7, 1 mM Na3VO4, 1% Triton X-100, pH 7.2 supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich). The frozen brains were homogenized in the same buffer and centrifuged at 12,000 x g for 20 min at 4°C. Protein concentration was quantified by using the Bio-Rad DC™ protein assay kit following the manufacturer’s instructions (Bio-Rad). Equal amounts of protein were separated on either 12% or 15% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, Dutscher, Brumath, France). After blocking, membranes were probed with the following antibodies: monoclonal anti-APP N-terminal (22C11, 1/500, Millipore, Molsheim, France), monoclonal anti-Aβ (6E10, 1/500, Covance), polyclonal anti-APP C-terminal (APP-CTF, 1/2000, Sigma-Aldrich), monoclonal anti-HIF-1α (1/500; R&D Systems Europe, Bio-Techne, Lille, France) and monoclonal anti-actin (1/5000, Sigma-Aldrich). Membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary IgG antibodies (Jackson Immunoresearch, West Grove, PA, USA) immunoblot signals were visualized with ECL chemiluminescence kit (GE Healthcare). Quantitative assessments were obtained by densitometric scanning using ImageJ software.
Horseradish peroxidase conjugated secondary igg antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase-conjugated secondary IgG antibodies are a type of laboratory reagent used in various immunoassay techniques. They consist of secondary antibodies that are chemically linked to the enzyme horseradish peroxidase. This enzyme can be used to catalyze a colorimetric or chemiluminescent reaction, allowing for the detection and visualization of target proteins in samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Monoclonal anti actin, by Merck Group (1 mentions)
Ecl chemiluminescence kit, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Monoclonal anti aβ 6e10, by Fortrea (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Dmem f12 medium, by Corning (1 mentions)
Rabbit anti gfp polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti ubiquitin antibody p4d1, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated goat anti mouse secondary fab, by Jackson ImmunoResearch (1 mentions)
Rabbit polyclonal anti cleaved caspase 3 asp175 antibody, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti β tubulin, by Merck Group (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti hemagglutinin ha antibody, by Fortrea (1 mentions)
Trypsin edta, by Wisent (1 mentions)
Penicillin streptomycin, by Wisent (1 mentions)
Protein g coupled dynabeads, by Thermo Fisher Scientific (1 mentions)
Rat anti ha tag, by Roche (1 mentions)
P jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
5 protocols using horseradish peroxidase conjugated secondary igg antibodies
1
Astrocyte Protein Extraction and Western Blot
2
Antibody Sources for Western Blotting
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Mouse monoclonal anti-hemagglutinin (HA) antibody was purchased from Covance Inc. (Berkeley, CA); rabbit polyclonal anti-HA, monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monoclonal anti-GFP antibodies were obtained from Abcam Inc. (Cambridge, MA); mouse monoclonal anti-Flag M2 antibody was from Sigma; rabbit polyclonal anti-GFP antibody was from Life Technologies. Mouse monoclonal anti-human transferrin antibody was from Invitrogen. Mouse monoclonal anti-β-tubulin was from Sigma. Mouse monoclonal anti-ubiquitin antibody (P4D1) was from Santa Cruz Biotechnology. Rabbit polyclonal anti-cleaved caspase-3 (Asp175) antibody was purchased from Cell Signaling Technology (kindly provided by Dr. Peter Siegel, McGill University). Horseradish peroxidase-conjugated secondary IgG antibodies, as well as FITC-conjugated goat anti-mouse secondary Fab were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All Alexa Fluor® conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). Alpha-minimum essential medium (α-MEM), fetal bovine serum, penicillin/streptomycin, and trypsin-EDTA were purchased from Wisent (Saint-Bruno, QC, Canada). The DMEM/F12 medium was from Corning. All other chemical and reagents were obtained from BioShop Canada (Burlington, ON, Canada), Sigma or Fisher Scientific and were of the highest grade available.
3
Immunoprecipitation and Western Blot Analysis
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Gene description Probes ID For the immunoprecipitation (IP) assay, 250 μg of protein (cell lysates) in 500 μL of RIPA buffer was incubated overnight at 4°C with unspecific mouse/rabbit/rat IgG antibodies (Jackson Immunoresearch) or rat anti-HA-tag (Roche Diagnostics) or mouse anti-FlagM2 and rabbit anti-APP-CTF (Sigma-Aldrich) at 4 ng/μL and then pulled-down for 2 hours using 50 μL of protein G-coupled dynabeads (Thermo Fisher Scientific). After washing three times with 0.3 M NaCl solution, samples were denatured and subjected to western blot (WB) using APP-CTF, anti-FlagM2, or anti-HA antibodies, and the corresponding horseradish peroxidase-conjugated secondary IgG antibodies (Jackson Immunoresearch). The EasyBlocker kit was used to limit unspecific signal according to the manufacturer's recommendations (GeneTex, Euromedex, Souffelweyersheim, France).
4
Antibody Panel for Stress Response
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Antibodies for IRE1α (#3294), PDI (#3501), PERK (#3192), eIF2α (#5324) and its phosphorylated form at ser51 (#3398, p-eIF2αser51) GRP78 (used in the cancer experiment, #3177), P62 (#5114), Bcl-2 (#3498), p-ULK1ser757 (#14202), ULK1 (#8054), p-JNKThr183/Tyr185 (#4668), JNK (#9252), ATF4 (#11815), Beclin-1 (#3495), caspase 12 (#2202), and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology. GAPDH (ab9485) antibody was purchased from Abcam (Cambridge, United Kingdom). LC3I and LC3II were measured by antibody (L7543) that was purchased from Sigma-Aldrich (St. Louis, MO, United States). Monoclonal primary antibodies were used for the detection of heat shock protein 70 (HSP70, StressGen, SPA-810), heat shock protein 60 (HSP60, StressGen, SPA-806), heat shock protein 90 (HSP90, StressGen, SPA-835), heat shock protein 47 (HSP47, StressGen, SPA-470) and thioredoxin interacting protein (TXNIP and MBL). Polyclonal primary antibodies were used to detect thioredoxin (TRX, IMCO, and ATRX-06), actin (Sigma, A-2066), heat shock protein 25 (HSP25, StressGen, and SPA-801), glucose-regulated protein 78 (GRP78, StressGen, SPA-826, used in the acute experiments). Horseradish peroxidase conjugated IgG secondary antibodies were used (Jackson ImmunoResearch Laboratories, PA, United States and StressGen and Zymed, San Francisco, CA, United States).
5
Western Blot Analysis of Apoptotic Proteins
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Proteins were extracted from cells of each group and their concentration was determined by BCA Kit (Biyuntian Biotech Company, China) according to instructions. The protein extracts were added with loading buffer and boiled for 10 min at 95 °C. Each well was loaded with 30 μg of samples. Then, 10% polyacrylamide gel electrophoresis (PAGE) was used to isolate proteins before polyvinylidene fluoride (PVDF) (Millipore, USA) transmembrane and 1 hr of closing with 5% bovine serum albumin (BSA) at room temperature. Later, primary antibodies BAK1 (ab104124, Abcam Company), caspase-3 (ab13586, Abcam Company), Bax (ab32503, Abcam Company), Bcl-2 (ab32124, Abcam Company), and β-actin (ab8226, Abcam Company) were added respectively for overnight incubation at 4 °C. Later, after washing with TBST (Tris-buffered saline with Tween 20) for 5 min×3 times, horseradish peroxidase- conjugated IgG secondary antibodies (Jackson Labs) were added for 1 hr of incubation at room temperature. Finally, the proteins were visualized by the chemilu -minescence reagent and analyzed Bio-rad Gel Dol EZ imager (GEL DOC EZ IMAGER, Bio-rad, California, USA). Grey value analysis of the target band was analyzed using software image J. We took β-actin as the internal reference gene.
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