The total protein content was extracted from pulp tissue and HDPFs by using lysis buffer containing protease inhibitors (Sigma-Aldrich, USA). The protein concentration was measured by using a BCA-200 protein assay kit (Pierce, Rockford, Ill., USA). Equal amounts of protein were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked in TRIS-buffered saline with Tween (TBST) containing 5 % nonfat dry milk for 2 h. For the detection of caspase-1, the membranes were probed overnight at 4 °C with a rabbit anti-human caspase-1 antibody diluted 1:100 (Abcam, US) and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-rabbit IgG antibody diluted 1:10,000 (Santa Cruz Biotechnology, Calif., USA). For the detection of NLRP3, the membranes were incubated overnight at 4 °C with a rabbit anti-human NLRP3 antibody diluted 1:100 (Abcam, USA) and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-rabbit IgG antibody diluted 1:10,000 (Santa Cruz Biotechnology). Protein bands were visualized on X-ray film by using an enhance chemiluminescence system (GE Healthcare, Buckinghamshire, UK). The relative protein expression intensities were quantified by densitometry by using Quantity One analysis software.
Horseradish peroxidase conjugated anti rabbit igg antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit IgG antibody is a secondary antibody that binds to rabbit immunoglobulin G (IgG) antibodies. The horseradish peroxidase (HRP) enzyme is conjugated to the antibody, allowing for detection and visualization of target proteins in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated anti mouse igg antibody, by Santa Cruz Biotechnology (4 mentions)
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Chemidoc apparatus, by Bio-Rad (2 mentions)
Ecl detection system, by Roche (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (2 mentions)
Anti goat igg horseradish peroxidase conjugated antibodies, by Santa Cruz Biotechnology (2 mentions)
Ecl prime kit, by Cytiva (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Quantity one program, by Bio-Rad (1 mentions)
Enhance chemiluminescence system, by GE Healthcare (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ripa lysate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti nestin, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti vegf, by Novus Biologicals (1 mentions)
Anti bfgf, by Santa Cruz Biotechnology (1 mentions)
Anti sox2, by Santa Cruz Biotechnology (1 mentions)
19 protocols using horseradish peroxidase conjugated anti rabbit igg antibody
1
Quantification of Caspase-1 and NLRP3 Proteins
2
Western Blot Analysis of Cellular Proteins
3
Quantifying Muscle Protein Expression
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The presence of various proteins was quantitated by performing western blot analysis. The gastrocnemius muscles and soleus of the mice were homogenized in the above-mentioned lysis buffer. Approximately 30 μg of protein was added to each lane of a 10% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked with 5% bovine serum albumin at room temperature for 1 h and incubated with primary antibodies at 4°C overnight, followed by the corresponding secondary antibodies. All primary antibodies were sourced from Cell Signaling Technology (Danvers, MA, USA) and horseradish peroxidase–conjugated anti-rabbit IgG antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were used as secondary antibodies. ECL Western blotting detection reagent (GE Healthcare, Little Chalfont, UK) was used to image the protein bands which were visualized on a WSE-6100 LuminoGraph system (ATTO, Tokyo, Japan) and quantified using the Quantity One program (Bio-Rad).
4
Western Blot Analysis of Kidney Proteins
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Protein from collected kidney tissues and HK-2 cells were extracted using RIPA lysate (Thermo Fisher Scientific). The total protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Samples with equal amounts of protein were separated electrophoretically by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Following blocked with 5% skim milk powder at room temperature for 2 h, the membranes were incubated with indicated antibodies including phosphorylated IκBα (p-IκBα), SIRT1, and inducible nitric oxide synthase (iNOS) (1:1000; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) at 4 °C overnight. β-actin was used as a loading control. All the blots were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology) for 1 h. Finally, the proteins were detected by an enhanced chemiluminescent detective system (Amersham Biosciences UK Ltd., Little Chalfont, UK).
5
Protein Expression Analysis in bEnd.3 and NSCs
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To confirm the difference in expressed proteins between the bEnd.3 cells and NSCs, proteins were extracted from ipsilesional brain tissue using a RIPA lysis buffer (Thermo Fisher, USA). For western blotting, equal amounts (30 μg) of protein extracts in a lysis buffer were used in a 12% SDS-PAGE analysis and transferred onto the polyvinylidene fluoride membranes. A 5% skimmed milk solution was used to block the membrane for 1 h at room temperature on a rocker. The housekeeping gene, β-actin, was employed as a loading control. Anti-Sox2 (1:1000, Santa Cruz, USA), anti-Nestin (1:1000, Abcam, UK), anti-VEGF (1:1000, Novus, USA), anti-bFGF (1:1000, Santa Cruz, USA), anti-CD31 (1:000, Abcam, UK), and anti-β-actin (1:1000, Santa Cruz, USA) antibodies were incubated with the membranes at 4°C overnight. After washing the membranes with TBST buffer, a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz, USA) at a dilution of 1:20,000 or an anti-mouse IgG antibody (KPL, Inc., Gaithersburg, MD, USA) at a dilution of 1:10,000 was added to the corresponding primary antibodies, followed by incubation for 1 h at room temperature. Bands were detected using ECL reagent (Millipore, USA).
6
Western Blot Analysis of URG4 Expression
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Cells at 70% to 80% confluence were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice in radioimmunoprecipitation assay buffer (RIPA; Cell
Signaling Technology, Danvers, MA) containing complete protease inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany). Fresh tissue samples were ground to powder in liquid nitrogen and lysed using SDS-PAGE sample buffer. Equal concentrations of each protein sample (20 μg) were separated on 6% SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with anti-upregulator of cell proliferation 4 antibody (1:1000, Sigma, HPA020134) overnight at 4°C. URG4 expression was determined using a horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:3000, Santa Cruz, SC-2004) and enhanced chemiluminescence (Pierce) according to the manufacturer’s protocols. The membranes were probed with anti-α-tubulin mouse monoclonal antibody (1:1000, Sigma, T5168) as a loading control.
Signaling Technology, Danvers, MA) containing complete protease inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany). Fresh tissue samples were ground to powder in liquid nitrogen and lysed using SDS-PAGE sample buffer. Equal concentrations of each protein sample (20 μg) were separated on 6% SDS polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with anti-upregulator of cell proliferation 4 antibody (1:1000, Sigma, HPA020134) overnight at 4°C. URG4 expression was determined using a horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:3000, Santa Cruz, SC-2004) and enhanced chemiluminescence (Pierce) according to the manufacturer’s protocols. The membranes were probed with anti-α-tubulin mouse monoclonal antibody (1:1000, Sigma, T5168) as a loading control.
7
Western Blot Analysis of GGT in Immune Cells and Exosomes
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For western blot determinations of GGT, isolated monocytes, lymphocytes, platelets and cultured endothelial HMEC-1 cells—harvested in hypotonic lysis buffer (10 mM Tris–HCl, pH 7.8)—or plaque derived exosomes were used. Non-adherent lymphocytes were isolated from plated PBMCs, whereas platelets were obtained from buffy-coat derived platelet concentrates. All samples were thoroughly washed in order to remove contaminating plasma GGT. Endothelial HMEC-1 cells were grown in MCDB 131 medium (Life Technologies) supplemented with 1 µg/ml hydrocortisone, 10 ng/ml EGF (Life Technologies) and 10 % v/v fetal calf serum, and cultured at 37 °C in a 5 %/95 % CO2/air atmosphere.
All samples were separated by 8 % SDS-PAGE [37 (link)] and incubated with rabbit anti-GGT IgG directed against the C-terminal 20 amino acids of human GGT heavy chain prepared as described [36 (link)]. Visualization of protein bands was obtained using a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ECL detection system (Roche, Basel, Switzerland). Bands were analyzed with a Bio-Rad ChemiDoc apparatus equipped with the QuantityOne software.
All samples were separated by 8 % SDS-PAGE [37 (link)] and incubated with rabbit anti-GGT IgG directed against the C-terminal 20 amino acids of human GGT heavy chain prepared as described [36 (link)]. Visualization of protein bands was obtained using a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ECL detection system (Roche, Basel, Switzerland). Bands were analyzed with a Bio-Rad ChemiDoc apparatus equipped with the QuantityOne software.
8
Antibody-based Immunoblotting Assays
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Antibodies against the following proteins were obtained from Cell Signaling Technology (Danvers, MA, USA): p38 (cat. #9212; 1:1,000 dilution), phospho-p38 (#4511; 1:1,000 dilution), Jun N-terminal kinase (JNK, #9252; 1:1,000 dilution), phospho-JNK (#9251; 1:1,000 dilution), extracellular signal-related kinase (ERK, #4696; 1:1,000 dilution), phospho-ERK (#4370; 1:1,000 dilution), c-Fos (#4384; 1:1,000 dilution), phospho-c-Fos (#5348; 1:1,000 dilution), c-Jun (#9165; 1:1,000 dilution), phospho-c-Jun (#9164; 1:1,000 dilution), SMAD family members 2 and 3 (SMAD2/3, #5678; 1:1,000 dilution), phospho-SMAD2/3 (#8828; 1:1,000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, #2118; 1:5,000 dilution), and tubulin (#2148; 1:5,000 dilution). A horseradish peroxidase–conjugated anti-rabbit IgG antibody (1:5,000 dilution) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) was used as secondary antibody. DMSO (cat. #D2650), an MTT solution (#M2128), and α-ionone (#W259403) were provided by Sigma-Aldrich (St. Louis, MO, USA).
9
Western Blot Analysis of B3GNT3
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Total protein was prepared using the cell total protein extraction kits according to the manufacturer’s instruction (Millipore, Bedford, MA). Equal amounts of each protein extract (30 μg) were electrophoretically separated on 9.5% SDS polyacrylamide gels. Following transfer to polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA), membranes were blocked for 1 h at room temperature with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). The membranes were then incubated with anti-B3GNT3 antibody (1:1000, Proteintech, 18098-1-AP) overnight at 4°C. Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2000, Santa Cruz, SC-2004) was used to determine B3GNT3 expression. Finally, B3GNT3 expression was detected using ECL prime Western blotting detection reagent (Amersham) according to the manufacturer’s instructions. Blotted membranes were stripped and re-probed with an anti-Alpha-Tubulin antibody (Sigma, Saint Louis, MO) as a loading control.
10
Protein Expression Analysis in Cultured Cells
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Cells (2×105 cells/ml) were seeded in a 60 mm dish and cultured to 80% confluence. After starvation, cells were treated with MVE. Cells were then lysed with 1xRIPA buffer (50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% Triton-X100, pH 7.4, 1% SDS, 50 mM NaF, 1 mM Na3VO4, 5 mM Dithiothreitol, 1 mg/ml Leupeptin, and 1mM phenylmethylsulfonyl fluoride). Sample protein (40 μg) was separated in 10–12% SDS-polyacrylamide gels by electrophoresis. Proteins were transferred to PVDF membranes and incubated with antibodies to collagen (1:1000 rabbit source, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and matrix metalloproteinase 1 (MMP1, 1:1000 rabbit source, Santa Cruz Biotechnology). Membranes were then washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology). Blots were reacted with western reagent (ECL; Millipore Billerica, Ann Arbor, MA, USA) and exposed to X-ray film.
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