Antibodies against MAT1, MAT2A, Nrf2, p65, c-Jun, c-Fos, Jun B, Jun D, PTEN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for phosphorylated Akt and Akt were obtained from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-conjugated donkey anti-rabbit, anti-goat IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were acquired from Jackson Immunoresearch Laboratories (West Grove, PA). Anti-actin antibody and most of the reagents used for molecular studies were obtained from Sigma (St. Louis, MO). The MAT2A-luc reporter plasmid was kindly donated by Dr. SC Lu (University of Southern California, CA). An overexpression plasmid for inhibitor of κBα (IκBα) and a pGL-ARE minimal reporter (containing three copies of the ARE region of the quinone oxidoreductase promoter) were donated by Dr. KY Lee (Chonnam National University, Korea) and Dr. MK Kwak (Catholic University, Korea), respectively. pNF-κB-luc and pAP-1-luc reporter plasmids were purchased from Stratagene (La Jolla, CA). Mimic hsa-miR-146b-3p (Dharmacon, C30109201), has-miR-146a (Dharmacon, C30063003) or mimic negative control (Dharmacon, CN0010000105) were supplied from Thermo Fisher Scientific (Lafayette, CO).
Horseradish peroxidase conjugated donkey anti rabbit
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase-conjugated donkey anti-rabbit is a secondary antibody conjugated to the enzyme horseradish peroxidase. It is designed to detect and bind to rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Anti actin antibody, by Merck Group (1 mentions)
Alkaline phosphatase conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
C jun, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Sox30 rabbit polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Sox30, by Santa Cruz Biotechnology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Oxphos antibody cocktail, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti igf 1r, by Cell Signaling Technology (1 mentions)
Nupage lds sample buffer 4x, by Thermo Fisher Scientific (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Anti tom20, by Cell Signaling Technology (1 mentions)
Anti fak, by Cell Signaling Technology (1 mentions)
Anti vdac, by Thermo Fisher Scientific (1 mentions)
Anti pi3k, by Cell Signaling Technology (1 mentions)
Anti vinculin, by Cell Signaling Technology (1 mentions)
4 protocols using horseradish peroxidase conjugated donkey anti rabbit
1
Molecular Signaling Pathway Analysis
2
Immunohistochemical and Immunoblotting Analysis of SOX30 and Cell Cycle Regulators
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Immunohistochemical staining was performed using the antibody against SOX30 and Ki67 (1:100 or 1:50; Santa Cruz Biotechnology, Heidelberg, Germany) as described previously.40 (link)Fifty micrograms of protein was run on 10–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA, USA). After blocking with 5% milk for 2 h at room temperature, membranes was incubated overnight at 4 °C with primary antibodies. After incubation with the secondary antibody, the proteins were detected by chemiluminescence (Pierce, Rockford, IL, USA). The same membrane was stripped and incubated with ACTIN monoclonal antibody (1:2000; Sigma), serving as an internal control. Primary antibodies were SOX30 rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology), p53 mouse polyclonal antibody (1:1000; Santa Cruz Biotechnology), BAX rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology), P21 rabbit polyclonal antibody (1:1000; Cell Signaling Technology, Boston, MA, USA) and PMAIP1 (NOXA) rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugated donkey anti-rabbit and horseradish peroxidase-conjugated rabbit anti-mouse (1:3000, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).
3
Western Blot Analysis of Muscle Proteins
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Proteins from GC muscles of WT and NRKdKO animals were extracted in RIPA lysis and extraction buffer (Thermo Fisher Scientific, #89901) supplemented with protease (Sigma, #S8820) and phosphatase inhibitor cocktail (Sigma, #4906845001). Protein concentration was determined using a BCA assay (Thermo Fischer Scientific, #23227). Samples were first diluted to 1.5 mg/ml and boiled 5 min in NuPAGE™ LDS Sample Buffer (4X) (Invitrogen, #NP0007), run on 4–12% Bis-Tris Protein gels (Thermo Fischer Scientific, #BN1003), and transferred using the semi dry system from Life Technologies. Membranes were incubated overnight at 4°C with primary antibodies anti-NRK1 and anti-NRK2 (Ratajczak et al., 2016 (link)), anti-NAMPT from Bethyl, OxPhos antibody cocktail from Invitrogen, anti-VDAC, anti-TOM20, anti-IGF1R, anti-PI3K, anti-phospho-Akt (Ser473), anti-Akt, anti-Raptor, anti-FAK, and anti-Vinculin from Cell Signaling. Membranes were then washed and incubated for 1 h with horseradish peroxidase–conjugated donkey anti-rabbit (Jackson ImmunoResearch, #711-035-152, 1/5000). Proteins were visualized with chemiluminescent western blotting substrate (Thermo Fisher Scientific, #32132) using Amersham Hyperfilm™ films. Densitometry analysis was performed using Fiji. Protein levels in each lane were normalized to the levels of Vinculin as a loading control.
4
Western Blot Antibody Source Protocol
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Horseradish peroxidase‐conjugated donkey anti‐rabbit and goat anti‐mouse IgG secondary antibodies were from Jackson ImmunoResearch Laboratories (Westgrove,PA). Molecular weight marker, reagents and nitrocellulose membranes for SDS‐PAGE were acquired from Bio‐Rad (Mississauga, ON). Western lightning Plus‐ELC (PerkinElmer, 105001EA). Antibodies for GAPDH (#2118S), BACE1 (#5606P), AMPK (#2793S), pAMPK (#2531S), AKT (#4685S), pAKT S473 (#4058S), pAKT Thr172 (#2531S), ERK 1/2 (#4695S), pERK 1/2 (#9101S), p38 (#9212S), p‐p38 (#9211S), JNK (#9252S), pJNK (#4671S), GSK‐3β (#9315S) and pGSK‐3β (#5558S) were from Cell Signaling (Danvers, MA, USA) and Vinculin (#05‐386) was from Milipore. All other sources are listed throughout the text.
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