Indicated samples were loaded on 4% to 20% Novex gels (Cat# EC60285BOX; Thermo Fisher Scientific, Waltham, MA). Proteins were transferred at 120 V for 1 hour using TGS buffer (25-mM Tris pH = 8.5, 192-mM glycine, 0.1% (mass/vol) SDS), 20% (vol/vol) methanol as transfer buffer to 0.45-μm polyvinylidene difluoride membranes (Cat# IPVH00010; Millipore, Billerica, MA), preactivated in 100% methanol. A blocking step was then performed in TBST (50-mM Tris-HCl, pH 7.4, 150-mM NaCl, 0.1% Tween 20), 5% (mass/vol) nonfat dry milk for 1 hour at room temperature, then incubated separately with primary antibodies: CaV2.2 (Cat# TA308673; Origene, Rockville, MD), PSD95 (Cat# MA1–045, Thermo Fisher scientific), synaptophysin (Cat# MAB5258, Thermo Fisher scientific, San Diego, CA), or flotillin (Cat# F1180; Sigma, St. Louis, MO) in TBST, 5% (mass/vol) BSA, at 4°C overnight. After incubation in horseradish peroxidase–conjugated (HRP) secondary antibodies (Jackson Labs) revelation was performed using enhanced luminescence (WBKLS0500; Millipore) before exposure to photographic film. Films were scanned and quantified using Un-Scan-It gel scanning software (version 6.1; Silk Scientific Inc).
Secondary horseradish peroxidase hrp conjugated antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Secondary horseradish peroxidase (HRP)-conjugated antibodies are laboratory reagents used for the detection and quantification of target proteins or antigens in various immunoassays. These conjugated antibodies bind to the primary antibody that recognizes the target, and the HRP enzyme catalyzes a color-producing reaction, enabling visualization and measurement of the target.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved parp, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Flotillin, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Novex gel, by Thermo Fisher Scientific (1 mentions)
Synaptophysin, by Thermo Fisher Scientific (1 mentions)
Psd 95, by Thermo Fisher Scientific (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Un scan it gel scanning software, by Silk Scientific (1 mentions)
Ikkα β sc 7607, by Santa Cruz Biotechnology (1 mentions)
Poly adp ribose polymerase parp 9542, by Cell Signaling Technology (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Dextran sulfate sodium salt, by Santa Cruz Biotechnology (1 mentions)
Rat anti ha 3f10 antibody, by Roche (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Westernsure ecl substrate, by LI COR (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
10 protocols using secondary horseradish peroxidase hrp conjugated antibodies
1
Western Blot Analysis of Neuronal Proteins
2
Western Blot Analysis of Inflammatory Markers
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Inducible nitric oxide synthase (iNOS; M00368) was used as a primary antibody from Boster Biological Technology (Pleasanton, CA, USA). Inhibitors of κB-α (IκB-α; sc-203), IκB kinase-α/β (IKK-α/β; sc-7607), HO-1 (sc-136960), and β-actin (sc-47778) were used as primary antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA). NF-κB p65 (4764), phospho-IκB-α (9246), phospho-IKK-α/β (Ser176/180; 2697), kelch-like ECH-associated protein 1 (keap1; sc-514914), and poly ADP-ribose polymerase (PARP; 9542) were used as primary antibodies (Cell Signaling Technology, Danvers, MA, USA). Nrf2 (ab92946) was used as a primary antibody (Abcam, Cambridge, UK). Horseradish peroxidase-conjugated (HRP) secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Commercial enzyme-linked immunosorbent assay (ELISA) kits for PGE2, myeloperoxidase (MPO), and SOD were purchased from R&D Systems (Minneapolis, MN, USA), Biovision (Milpitas, CA, USA), and Abcam (Cambridge, MA, USA). Unless otherwise specified, all remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Immunoblotting and Immunoprecipitation Analysis
4
Inflammasome Activation Assay Protocol
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ENU and EMS were purchased from Sigma-Aldrich. CellTiter 96 AQueous One Solution cell proliferation assay was obtained from Promega. Dextran sulfate sodium salt was obtained from Santa Cruz Biotechnology. LPS Escherichia coli O55:B5 (catalog no. 437625) was purchased from Calbiochem/EMD Biosciences. Caspase-1 inhibitor VX765 was purchased from Selleck Chemicals. NLRP3 inflammasome inhibitor MCC950 was purchased from AdipoGen Life Sciences, Inc. Nigericin (sodium salt) was purchased from MilliporeSigma. Rabbit anti-IL-1β (Ab9787) was purchased from Abcam. Rat anti-HA (3F10) antibody was obtained from Roche. Mouse anti-FLAG M2 antibody (F1804) was purchased from Sigma-Aldrich. Secondary horseradish peroxidase (HRP)-conjugated antibodies were purchased from Jackson ImmunoResearch. Alexa Fluor 448 and 594 secondary antibodies were obtained from Invitrogen.
5
Western Blot Analysis of Protein Expression
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Cells were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with a protease inhibitor cocktail (EMD Biosciences, CA) for 30 min and total protein was determined (Bradford; ThermoFisher). Cell lysates were boiled for 5 min at 95 °C, loaded to a sodium dodecyl sulfate-polyacrylamide gel (30 μg of total protein/lane) and after gel electrophoresis and protein transfer to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific), the membranes were blocked with 5% milk in Tris-buffered saline with 0.05% Tween-20 (TBS/T) for 1 h at room temperature. After 3 washes with TBS/T, the membranes were incubated with primary antibodies against myc-tag (Cell Signaling Technology) and β-actin (cat. no. A1978, Sigma) in a shaker at 4 °C overnight. Following 3 more TBS/T washes, secondary horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch Laboratories Inc.) were added for 1 h at room temperature. Membranes were washed 3 times and proteins were detected in a C-DiGit blot scanner after adding WesternSure ECL substrate (Li-Cor Biotechnology, Lincoln, NE).
6
Western Blot Analysis of TAp63, ΔNp63, and Cas9
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Cells were lysed in ice-cold M-PER lysis buffer purchased from Thermo Fisher Scientific. Cell lysates were clarified by centrifugation (20 min at 15,000 rpm and 4° C), and protein concentrations were determined using BCA protein assays (Thermo Fisher Scientific). Equal amounts of protein were separated using SDS-PAGE. The resolved proteins were then transferred to Hybond PVDF transfer membranes (Millipore, Bedford, MT, USA) and incubated with primary and secondary antibodies according to the Supersignal® West Pico chemiluminescence protocol (Thermo Fisher Scientific). Primary anti-TAp63 and anti-ΔNp63 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), and an anti-Cas9 antibody was obtained from Novus Biologicals, Inc. (Littleton, CO, USA). An anti-β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary horseradish peroxidase (HRP)-conjugated antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA, USA).
7
Western Blot Analysis of DNA Damage Signaling
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Cells were seeded 24 hours prior to treatment and incubated with the respective inhibitors for 2 hours or as otherwise indicated. Cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Western blotting was performed as described previously (16 (link)). Primary antibodies were obtained from Cell Signaling Technology against pAKT (S473; catalog no. 9271, RRID:AB_329825), pEGFR (Y1068; catalog no. 2234, RRID:AB_331701), Cleaved Caspase 3 (catalog no. 9661, RRID:AB_2341188), Cleaved PARP (catalog no. 9541, RRID:AB_331426), Phospho-Histone H2A.X (Ser139; catalog no. 9718, RRID:AB_2118009), pATM (S1981; catalog no. 5883, RRID:AB_10835213), pDNA PKcs (S2056; catalog no. 68716, RRID:AB_2939025), and pATR (S428; catalog no. 2853, RRID:AB_2290281). Secondary horseradish peroxidase (HRP)-conjugated antibodies were obtained from Jackson ImmunoResearch. ECL-Plus substrate (Bio-Rad) and Bio-Rad ChemiDoc MP imager were used according to the manufacturer's recommendations.
8
Western Blot Analysis of c-Myc Protein
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Cells were lysed in cell lysis buffer (Cell Signaling Technology, cat. no. 9803, Danvers, MA) supplemented with a protease inhibitor cocktail (Thermo Scientific, cat. no. 78425, Waltham, MA) and total protein was determined (ThermoFisher, cat. no. 23236, Waltham, MA). Cell lysates were boiled for 5 min at 95 °C, loaded to a polyacrylamide gel (30 μg of total protein/lane) and after gel electrophoresis and protein transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, cat. no. IPVH00010, Burlington, MA). The membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated with primary antibodies against the c-Myc epitope (Cell Signaling Technology, cat. no. 2278s, Danvers, MA) at 4 °C overnight, or with GAPDH (Sigma Aldrich, cat. no. G9545, St. Louis, MO) for 1 h at room temperature. Following 3 more TBST washes, secondary horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) were added for 1 h at room temperature. Membranes were washed 3 times, developed with Clarity Max Western ECL Substrate (Biorad, cat. no. 1705062, Hercules, CA) and detected in a C-DiGit blot scanner.
9
Western Blot Analysis of Apoptosis Markers
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Total protein was isolated using RIPA Lysis Buffer (Beyotime Biotechnology) and protein concentration was measured with a BCA protein assay kit (Beyotime Biotechnology). Forty microgram of protein was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred onto the polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 5% fat‐free milk solution containing 0.1% Tween‐20 for 1 hour at room temperature. Later, membranes were incubated at 4°C overnight with primary antibodies against HOXA10 (1:1000; GeneTex), GAPDH (1:5000; proteintech), and BCL2, Bax, cleaved Caspase‐9, cleaved Caspase‐3, and cleaved PARP (1:1000; Cell Signaling Technology). Subsequently, the membranes were incubated with HRP (horseradish peroxidase)‐conjugated secondary antibodies (1:10 000; Jackson ImmunoResearch Inc) for 1 hour at room temperature. After washing with TBST buffer, the targeted proteins were visualized with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore).
10
LPS-Induced Inflammation in C57BL/6 Mice
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C57BL/6 mice (20 ± 2 g, 6–8 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Animal care was performed according to protocols approved by the Animal Care and Use Committee in Zhejiang A & F University. LPS and streptomycin were purchased from Sigma-Aldrich (St.Louis, MO, USA). Antibodies specific to phospho-p38 MAPK, p38 MAPK, phospho-ERK1/2, ERK1/2, I-κBα and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, USA). HRP (horseradish peroxidase)-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA).
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