Trypsin and bafilomycin A1 were purchased from Sigma Chemical Co., (St. Louis, MO, USA). Anti-GAPDH monoclonal antibody from Calbiochem-Merck Chemicals Ltd. (Nottingham, UK). MnSOD, catalase and OGG-1 antibodies from Adipogen (San Diego, CA, USA). Phospho-mTOR, mTOR, phosphor-AKT, AKT, LC3, p62, LAMP-I, Cathepsin B, HexA and galectin-3 were obtained from Cell Signaling Technology. A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN, USA). Grace’s insect medium was purchased from Gibco. The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA, USA).
Immun star hrp substrate kit
Manufactured by Bio-Rad
Sourced in United States
The Immun Star HRP substrate kit is a chemiluminescent reagent designed for the detection of horseradish peroxidase (HRP) in western blotting applications. The kit provides a sensitive and reliable method for visualizing HRP-labeled target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Merck Group (8 mentions)
Anti actin monoclonal antibody, by Merck Group (4 mentions)
Prussian blue, by Merck Group (3 mentions)
Pdh activity kit, by Abcam (3 mentions)
Immobilon membranes, by Cytiva (3 mentions)
Anti lipoic acid, by Merck Group (3 mentions)
Hoechst 3342, by Thermo Fisher Scientific (3 mentions)
Anti pank3, by Thermo Fisher Scientific (3 mentions)
Grace s insect medium, by Thermo Fisher Scientific (2 mentions)
Anti pank2, by Abcam (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Oxyblot protein oxidation detection kit, by Merck Group (2 mentions)
Sudan black, by Merck Group (2 mentions)
Rabbit anti flag, by Merck Group (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Anti atg12, by Santa Cruz Biotechnology (2 mentions)
Anti sirt1, by Cell Signaling Technology (2 mentions)
Anti parkin, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
29 protocols using immun star hrp substrate kit
1
Biochemical Assay Reagents and Antibodies
2
Antibody-based Mitochondrial Protein Analysis
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Monoclonal anti-actin antibody, Prussian Blue, pantothenate, pantethine, vitamin E, L-carnitine, thiamine, Luperox® and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mitochondrial acyl carrier protein (mtACP), anti-NF-Y, anti-FOXN4 and anti-hnRNPA/B were purchased from Invitrogen/Molecular Probes (Eugene, OR). Anti-phospho-PGC1α was purchased from RD systems. NFS1 antibody and Omega 3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PANK2, anti-PGC1α, complex 1 activity kit and PDH activity kit were purchased from Abcam (Cambridge, UK). Anti-TFAM was purchased from Cell Signaling. BODIPY® 581/591 C11 was purchased from Thermo-Fisher (Waltham, MA). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).
3
Fibroblast Lysate Preparation and Western Blot Analysis
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Whole cellular lysate from fibroblasts was prepared by gentle shaking with a buffer containing 0.9% NaCl, 20 mM Tris-HCl, pH 7.6, 0.1% Triton X-100, 1 mM phenylmethylsulfonylfluoride and 0.01% leupeptin. The protein content was determined by the Bradford method. Electrophoresis was carried out in a 10–15% acrylamide SDS/PAGE and proteins were transferred to Immobilon membranes (Amersham Pharmacia, Piscataway, NJ, USA). Next, membranes were washed with PBS, blocked over night at 4 °C and incubated with the respective primary antibody solution (1:1000). Membranes were then probed with their respective secondary antibody (1:2500). Immunolabeled proteins were detected by chemiluminescence method (Immun Star HRP substrate kit, Bio-Rad Laboratories Inc., Hercules, CA, USA). Western blot images were quantified using ImageJ software.
4
Quantitative Western Blot Analysis
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Western blotting was performed using standard Methods. After transferring protein to a nitrocellulose membrane. The membrane was incubated with primary antibodies diluted 1:1000, and then with the corresponding secondary antibody coupled to horseradish peroxidase at a 1:2500 dilution. Specific protein complexes were identified by ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA) using the Immun Star HRP substrate kit (Biorad Laboratories Inc., Hercules, CA, USA). ImageLab™ version 5.0 software (Bio-Rad, Hercules, CA, USA) was used to analyze protein expression levels.
5
Western Blot Protein Analysis Protocol
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Whole cellular lysate from cells was prepared by gentle shaking with a lysis buffer containing 0.9% NaCl, 0.1% triton X−100, 20 mM Tris-ClH, pH 7.6, 1 mM phenylmethylsulfonylfluoride and 0.01% leupeptine. Electrophoresis was carried out in a 10–15% acrylamide SDS/PAGE and proteins were transferred to Immobilon membranes (Amersham Pharmacia, Piscataway, NJ, USA). After this, membranes were washed with PBS, blocked over night at 4 °C and incubated with primary antibodies at 1:1000. Then, membranes were incubated with secondary antibody (1:2500). Immunolabeled proteins were detected by chemiluminescence method (Immun Star HRP substrate kit, Bio-Rad Laboratories Inc., Hercules, CA, USA) and quantified using ImageJ software (see: http://rsb.info.nih.gov/ij/download.html ).
6
Western Blot Analysis of Protein Complexes
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Western blotting was performed using standard methods. After protein transfer, the membrane was incubated with various primary antibodies diluted at 1:1,000 and then with the corresponding secondary antibodies coupled to horseradish peroxidase at a 1:10,000 dilution. Specific protein complexes were identified using the Immun Star HRP substrate kit (Biorad Laboratories Inc.).
7
Mitochondrial Dysfunction Biomarker Assay
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Sudan Black, Prussian Blue, ( ±) α-LA, Luperox® DI (tert-Butyl peroxide), anti-fatty acid synthase (FAS), and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). BODIPY® 598/591 C11, MitoTracker Deep Red FM, DAPI, were purchased from Invitrogen/Molecular Probes (Eugene, OR). MitoPeDPP® was purchased from Dojindo Molecular Technologies, Inc. (Rockville,MD) Anti-PANK2, anti-MTND1, anti-NDUFA9, anti-NFS1, anti-ISCU, anti-LYRM4anti-NRF2, PDH hand complex I activity kit and aconitase kit were purchased from Abcam (Cambridge, UK), Anti-mitochondrial 10-formyltetrahydrofolate dehydrogenase (ALDH1L2), anti-alpha-aminoadipic semialdehyde synthase (AASS), anti-FOXN4, anti-hnRNPA/B,anti-NF-Y, anti-Tau, anti-GPX4 and anti-AASDHPPT were purchased from Thermo-Fisher (Waltham, MA). Anti-lipoic acid was acquired from Merck (Darmstadt, Germany). Anti-PLA2G6 and anti-SOD were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-actin was acquired from MyBiosource (San Diego, California, USA). OxyBlot Protein Oxidation Detection Kit was acquired from Merck (Darmstadt, Germany). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).
8
Western Blot Analysis of Protein Complexes
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Western blotting was performed using standard Methods described in previous manuscripts of the research group [8 (link)]. After protein transfer, membranes were incubated with various primary antibodies diluted 1:1000, and then with the corresponding secondary antibody coupled to horseradish peroxidase at a 1:10,000 dilution. Specific protein complexes were identified using the Immun Star HRP substrate kit (Biorad Laboratories Inc., Hercules, CA, USA).
Protein loading was assessed by Ponceau staining and actin expression levels. If the molecular weight of proteins did not interfere, membranes were re-probed with different antibodies. In the case of proteins with different molecular weights, membranes were cut and incubated with specific antibodies.
Protein loading was assessed by Ponceau staining and actin expression levels. If the molecular weight of proteins did not interfere, membranes were re-probed with different antibodies. In the case of proteins with different molecular weights, membranes were cut and incubated with specific antibodies.
9
Western Blot Analysis of Immunoprecipitation Samples
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Samples from the immunoprecipitation assays described above were separated by gel electrophoresis using 12% or 15% Tris-Tricine polyacrylamide gels, transferred to nitrocellulose membranes, and probed with rabbit anti-IdsD (1:2,000), rabbit anti-IdsE (1:2,000), rabbit anti-FLAG (1:4,000; Sigma-Aldrich, St. Louis, MO), rabbit anti-His6 (1:2,000; Abcam, Cambridge, England), or mouse anti-sigma-70 (1:1,000; Thermo, Fisher Scientific, Waltham, MA), followed by goat anti-rabbit conjugated to horseradish peroxidase (HRP) (1:5,000; KPL, Inc., Gaithersburg, MD) or goat anti-mouse conjugated to HRP (1:5,000; KPL, Inc., Gaithersburg, MD) and developed with Immun-Star HRP substrate kit (Bio-Rad Laboratories, Hercules, CA). Antibodies specific to IdsD amino acids 4 to 18 (EVNEKYLTPQERKAR) and IdsE amino acids 298 to 312 (EQILAKLDQEKEHHA) were raised in rabbits using standard protocols (Covance, Dedham, MA). Blots were visualized using a Chemidoc (Bio-Rad Laboratories, Hercules, CA). JPEG images of blots were converted to TIFF files using Adobe Photoshop (Adobe Systems, San Jose, CA), and figures were made in Adobe Illustrator (Adobe Systems, San Jose, CA).
10
Immunoblotting for Cellular Proteins
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The following antibodies and reagents were used: monoclonal anti-actin antibody and rabbit anti-VDAC1/Porin from Sigma-Aldrich (St. Louis, MO, USA); anti-MAP lc3-2b (N-20) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); a mixture of protease inhibitors from Boehringer Mannheim (Indianapolis, IN, USA); and Immun Star HRP substrate kit from Bio-Rad Laboratories (Hercules, CA, USA). Extra reagents were also obtained from Sigma-Aldrich.
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