Protein extraction was performed by using Cell Lytic buffer (Sigma-Aldrich) supplemented with a protease inhibitors cocktail (Sigma-Aldrich) plus phosphatases inhibitors (Na3VO4 1 mM; NaF 10 mM) and resolved by electrophoresis through NuPAGE Bis-Tris gel (Invitrogen) and electroblotted onto nitrocellulose (Protran, Sigma-Aldrich) membrane. Blots were incubated with indicated primary antibodies in 5% non-fat dry milk in PBS plus 0.1% Tween20 overnight at 4 °C. Primary antibodies were: anti-PERK (1:500; Cell Signaling; Danvers, MA, US), anti-ERK1/2 (1:500; Cell Signaling), anti-ERK1/2 (1:500; Cell Signaling), anti-Nrf2 (1:500; Genetex; Alton Pkwy Irvine, CA, US), anti-Herp (1:500; Sigma-Aldrich), anti-AKR1C1 (1:500; NOVUS), anti-AKR1C2 (1:500, Merck), anti-AKR1C3 (1:500; Cell Signaling), anti-Tubulin-α (1:5000; Santa Cruz Biotechnology; Santa Cruz, CA, US). Detection was achieved using horseradish peroxidase-conjugate secondary antibody (1:5000; Jackson ImmunoResearch; Cambridge, UK) and visualized with ECL plus (Amersham Biosciences; Amersham, UK). Images were acquired by using a ChemiDoc™ Touch Imaging System (Bio-Rad; Berkeley, CA, US) and analyzed by Image Lab software (Bio-Rad).
Horseradish peroxidase conjugate secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United Kingdom, United States
Horseradish peroxidase-conjugate secondary antibody is a laboratory reagent that binds to the Fc region of primary antibodies. It is used in various immunodetection techniques, such as Western blotting, ELISA, and immunohistochemistry, to amplify and visualize the target antigen signal.
Automatically generated - may contain errors
Lab products found in correlation
Protran, by Merck Group (4 mentions)
Cell lytic buffer, by Merck Group (4 mentions)
Image lab software, by Bio-Rad (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (3 mentions)
Protease inhibitors cocktail, by Merck Group (3 mentions)
Ecl plu, by Cytiva (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Hyperfilm ecl, by GE Healthcare (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti nrf2, by GeneTex (1 mentions)
Anti herp, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
12 protocols using horseradish peroxidase conjugate secondary antibody
1
Western Blot for Protein Analysis
2
Protein Detection via Western Blotting
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Cell extracts were centrifuged at 13 000 g for 10 min at 4 °C. Protein concentrations were determined with the Bio-Rad Protein Assay (Bio-rad Laboratories, Hercules, CA, USA). Cell extracts or immunoprecipitates were separated by SDS-PAGE and transferred onto nylon membranes (Immobilon P, Millipore, Bedford, MA, USA). Membranes were incubated with primary antibodies followed by horseradish peroxidase-conjugate secondary antibody (Jackson Laboratories, Ann Arbor, MI, USA) and visualized with ECL plus (Amersham Bioscience, Little Chalfont, UK).
3
Protein Extraction and Western Blot Analysis
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Total protein extraction was performed by homogenizing cells in Ripa lysis buffer containing 1X protease and phosphatase inhibitors cocktail (ThermoFisher Scientific, Waltham, MA, USA). The homogenates, after 30 min of incubation on ice, were then centrifuged at 13,000 rpm for 30 min at 4 °C. Protein concentrations were determined using the Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Lysates obtained from liver tissue were analyzed in denaturing condition through SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Bioscience, Little Chalfont, UK). Membranes were incubated with primary antibodies followed by horseradish peroxidase-conjugate secondary antibody (Jackson Laboratories, Ann Arbor, MI, USA) and visualized with ECL (Western nova 2.0, Cyanagen, Italy). Densitometric analysis of immunoblots was performed by ImageJ64 image processing software for electrophoresis gel analysis.
Primary antibodies, diluted according to the manufacturer’s instruction, were as follows: β-Actin (sc-47778, C4) and Beclin-1 (sc-48341, E-8) purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), p-mTOR (Ser2481) (2974) and p-Akt (Ser 473) (9271) p-AMPK T172 (40H9), p-ULK1 (Ser555) (D1H4) and ULK1 (D8H5) from Cell Signaling (Danvers, MA, USA), LC3B (ab51520) and AMPK (ab3759) from Abcam (Cambridge, UK), and p62/Sqstm1 (PB9444) from Boster (Pleasanton, CA, USA).
Primary antibodies, diluted according to the manufacturer’s instruction, were as follows: β-Actin (sc-47778, C4) and Beclin-1 (sc-48341, E-8) purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), p-mTOR (Ser2481) (2974) and p-Akt (Ser 473) (9271) p-AMPK T172 (40H9), p-ULK1 (Ser555) (D1H4) and ULK1 (D8H5) from Cell Signaling (Danvers, MA, USA), LC3B (ab51520) and AMPK (ab3759) from Abcam (Cambridge, UK), and p62/Sqstm1 (PB9444) from Boster (Pleasanton, CA, USA).
4
Protein Extraction and Western Blot Analysis
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Cells for each different experimental protocol were lysed in RIPA (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris pH 8.0, and 2 mM MgCl2). Protease and phosphatase inhibitors (protease inhibitor cocktail plus 5 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1 mM sodium molybdate, 50 mM 2-chloroacetamide, 2 mM 1,10-phenanthroline monohydrate, and 0,5 mM PMSF; Sigma-Aldrich) were added. Lysates were incubated at 4°C for 30 min. After centrifugation at 4°C for 10 min at 13,000 rpm to remove insoluble debris, the protein concentrations were determined using a Bradford assay with bovine serum albumin as the reference standard (Biorad). Equal amounts of protein (10 μg) were re-suspended in SDS-PAGE sample buffer. The samples were then separated on NuPAGE Bis-Tris gel (Life Technologies) and electroblotted onto nitrocellulose (Protran, Schleicher & Schuell) or PVDF (Millipore) membranes. Blots were incubated with primary antibody in 5% non-fat dry milk in PBS plus 0.1% Tween-20 overnight at 4°C. Detection was achieved using a horseradish peroxidase-conjugate secondary antibody (Jackson Immuno Research Laboratories) and visualised with ECL (GE Healthcare).
5
Quantitative Western Blot Analysis of Autophagy Markers
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Total proteins were extracted from PBMC, isolated by Ficoll-Hypaque isolated PBMC, by using the Cell Lytic buffer (Sigma-Aldrich, C3228) following addition of protease inhibitors and resolved by electrophoresis through NuPAGE Bis-Tris gel (Invitrogen, NP0321BOX) and electroblotted onto nitrocellulose (Protran, 10402062) or PVDF (Millipore, IPVH20200) membranes. Blots were incubated with indicated primary antibodies in 5% nonfat dry milk in PBS plus 0.1% Tween 20, overnight at 4 °C. Primary antibodies were: rabbit anti-LC3B (1:2000; Cell Signaling Technology, 2775), rabbit anti-SQSTM1 (1:2000; MBL, PM045) anti-GAPDH (1:60000; Calbiochem, CM1001). Detection was achieved using horseradish peroxidase-conjugate secondary antibody (1:5000; Jackson ImmunoResearch, 715-036-150) and visualized with ECL Prime (GE Healthcare, RPN2232) using ECL-Hyperfilm (GE Healthcare, 28-9068-40). Mouse anti-GAPDH antibody was used to monitor equal protein loading. Western blot images were analyzed densitometrically using a charge-coupled device camera (GelDoc 2000, Bio-Rad, Hercules, CA, USA) and processed with the QuantyOne software (Bio-Rad) in order to quantify the amount of LC3-II band intensity.
6
Huh7.5 Cells for HCV Cell Model
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The human hepatoma-derived cell line Huh7.5 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and 2 mM L-glutamine (Lonza).
J6/JFH1 cDNA (kindly provided by C. Rice, Rockefeller University) was used to generate the HCV cell model. Antibodies anti human KLF15, KLF3, TCF7L2 (Abcam), Adipsin, Adipogenin, Twist 1, beta Actin (Santa Cruz Biotechnology, Santa Cruz, CA), HCV NS5a (Austral Biologicals) were used for immunofluorescence assay. Detection was achieved using horseradish peroxidase–conjugate secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). ECL Plus Western and ECL-Hyperfilm (GE Healthcare Life Sciences) were used. Specific neutral lipids were stained with BODIPY (Invitrogen).
J6/JFH1 cDNA (kindly provided by C. Rice, Rockefeller University) was used to generate the HCV cell model. Antibodies anti human KLF15, KLF3, TCF7L2 (Abcam), Adipsin, Adipogenin, Twist 1, beta Actin (Santa Cruz Biotechnology, Santa Cruz, CA), HCV NS5a (Austral Biologicals) were used for immunofluorescence assay. Detection was achieved using horseradish peroxidase–conjugate secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). ECL Plus Western and ECL-Hyperfilm (GE Healthcare Life Sciences) were used. Specific neutral lipids were stained with BODIPY (Invitrogen).
7
Western Blot Analysis of Protein Targets
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Samples were separated on SDS-polyacrylamide gels and transferred to an Immobilon-P Membrane (IPVH00010, Millipore). After blocking for 1 h at room temperature with 5% no-fat milk (A0830, Applichem), the membranes were incubated with primary antibodies overnight at 4 °C. Antibodies for ARPC2 (HPA008352) were obtained from Atlas Antibodies. 14-3-3ε (CPCT-YWHAE-1), 14-3-3 α/β (CPCT-YWHAB-1) antibodies were obtained from DSHB, pRb antibody (554136) from BD-Pharmingen and vinculin antibody (ab11194) from Abcam, and the PAK4 pab 6508 were generated in our laboratory [31 (link)]. The membranes were then incubated with a horseradish peroxidase-conjugate secondary antibody (mouse: 715-035-150, Rabbit: 111-035-144, Jackson ImmunoResearch) for 1 h at room temperature. Membranes were developed by enhanced chemiluminescence (#32106, Thermo Science).
8
Protein Expression Analysis of Intestinal Samples
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The whole small intestine lysates were obtained by using the Cell Lytic buffer (Sigma-Aldrich) supplemented with a protease inhibitors cocktail (Sigma-Aldrich) plus phosphatases inhibitors (Na3VO4 1 mM; NaF 10 mM), and resolved by electrophoresis through SDS-PAGE, and electroblotted onto nitrocellulose (Protran, Sigma-Aldrich) membranes. Membranes were incubated with indicated primary antibodies in 5% non-fat dry milk (Bio-Rad) in PBS plus 0.1% Tween20 overnight at 4 °C. Primary antibodies were: anti-CFTR (clone CF3, ab278, Abcam, Cambridge, UK) 1:500, anti-TG2 (clone CUB7402, NeoMarkers, Invitrogen) 1:500, and anti-tubulin (Santa Cruz, CA, USA) 1:5000. Detection was achieved using horseradish peroxidase-conjugate secondary antibody (1:5000; Jackson ImmunoResearch; Cambridge, UK) and visualized with ECL plus (Amersham Biosciences Corp., Little Chalfont, UK). Images were acquired by using a ChemiDoc™ Touch Imaging System (Bio-Rad) and analysed by Image Lab software (Bio-Rad), as previously described16 (link).
9
Protein Extraction and Western Blotting
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For protein extraction, cells were suspended in RIPA lyses buffer plus protease and phosphatase inhibitors (Sigma-Aldrich). The protein extract was analyzed through SDS–PAGE and probed with different primary antibodies and horseradish peroxidase–conjugate secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and detected by ECL plus (GE Healthcare).
10
Western Blot Analysis of Small Intestine Proteins
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The whole small intestine lysates were obtained using the Cell Lytic buffer (Sigma) supplemented with a protease inhibitors cocktail (Sigma) plus phosphatases inhibitors (Na3VO4 1 mM; NaF 10 mM), resolved by electrophoresis through SDS-PAGE, and electroblotted onto nitrocellulose (Protran, Sigma) membranes. Membranes were incubated with indicated primary antibodies in 5% nonfat dry milk (Bio-Rad) in PBS plus 0.1% Tween20 overnight at 4 °C. Primary antibodies were: Anti-CFTR (clone M3A7 Abcam ab4067) 1:500, anti-TG2 (NeoMarkers) 1:750, and anti-βActin (Cell Signaling) 1:2000. Detection was achieved using horseradish peroxidase-conjugate secondary antibody (1:5000; Jackson ImmunoResearch; Cambridge, UK) and visualized with ECL plus (Amersham Biosciences; Amersham). Images were acquired using a ChemiDoc™ Touch Imaging System (Bio-Rad) and analyzed by Image Lab software (Bio-Rad), as previously described.
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