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Horseradish peroxidase hrp conjugated antibody

Manufactured by Santa Cruz Biotechnology
Sourced in United States

Horseradish peroxidase (HRP)-conjugated antibody is a laboratory reagent used in various immunoassays and immunochemical techniques. HRP is an enzyme that catalyzes the oxidation of substrates, producing a colored or luminescent signal that can be detected and quantified. The HRP-conjugated antibody binds to a specific target antigen, allowing the detection and visualization of the target in the sample.

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18 protocols using horseradish peroxidase hrp conjugated antibody

1

Antibody detection and analysis protocol

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The antibodies used in this study and their sources are as follows: Anti-GFP (11814460001, Roche; A6455, Invitrogen); Anti-phosphotyrosine-biotin (4G10-biotin; Upstate), Anti-SHP-1 (610126, BD Transduction Laboratories; 07-419, Upstate), Anti-HLA-C (F4/326 (IgG2a), a gift from S.Y. Yang (Memorial Sloan-Kettering Cancer Center, New York). The horseradish peroxidase (HRP) conjugated antibodies were from Santa Cruz. Streptavidin-HRP antibody was obtained from GE Healthcare. Allophycocyanin (APC)-conjugated anti-KIR2DL1 antibody used in flow cytometer studies was from Beckman Coulter (A22332).
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2

SDS-PAGE and Immunoblotting Analysis of Viral Particles

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Analysis of the particle distribution after ultracentrifugation was carried out by SDS-PAGE using 4-to-12% gradient Bis-Tris gels in morpholineethanesulfonic acid (MES)-SDS running buffer. After electrophoresis, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane by electroblotting, and subsequently, the membranes were blocked overnight at 4°C with 3% nonfat milk in TBST (Tris-buffered saline, 0.1% [vol/vol] Tween 20). After blocking, the membranes were incubated for 1 h at RT with anti-sHBsAg pAb (OriGene) or mAb AP33 diluted in 0.3% nonfat milk in TBST, washed with TBST, and then incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies (Santa Cruz Biotechnology). The results were developed using substrate for enhanced chemiluminescence (Thermo Scientific).
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3

Quantitative Western Blot Analysis of WT1

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G401 cells were washed with 800 µl 1X PBS 3 times for 5 min and lysed with 150 µl radioimmunoprecipitation assay per well. For kidney samples, tissue was homogenized in lysis buffer and sonicated for 3 min. The protein concentration of each sample was determined by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Proteins (50 µg/lane) were segregated using 10% SDS-PAGE gel and immunoblotting was performed using polyclonal antibodies against WT1 (AB10840; 1:2,500; Abcam, Cambridge, UK) and β-tubulin (AB6040; 1:5,000; Abcam). The two antibodies were used at 4°C overnight at 1 mg/ml in PBS with 5% non-fat milk according to the manufacturer's protocol. Then the PVDF membrane was probed with horseradish peroxidase (HRP)-conjugated antibodies (1:4,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Finally, immunoreactivity was detected using Chemiluminescent HRP Substrate reagent (Merck Millipore, Darmstadt, Germany) and the signal was analyzed using Bio-Rad ChemiDoc XRS+ (BioRad Laboratories, Inc., Hercules, CA, USA). β-tubulin was used as an internal control. Western blot analysis was performed in triplicate for each group.
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4

Protein Extraction and Western Blot Analysis

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Cells were lysed in ice-cold RIPA cell buffer (Sigma) supplemented with protease inhibitors (Sigma). The proteins were separated with a 4–12% PAGE gel and electrotransferred onto a PVDF membrane. The membrane was probed with primary antibodies and subsequently detected by horseradish-peroxidase (HRP) conjugated antibodies (Santa Cruz). The primary antibodies were β-catenin (Santa Cruz), Flag antibody (Sigma), HIF1α (Santa Cruz) and HIF2α (Santa Cruz).
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5

Oxidative Stress and Osteogenesis Regulation

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dl-Homocysteine, NaHS, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). TRIzol for RNA isolation was obtained from Thermo Fisher Scientific (Waltham, MA). Primary antibodies against heme oxygenase-1 (HO-1), S-adenosylhomocysteine, superoxide dismutase 2 (SOD-2), catalase-1, NADPH oxidase 2 (NOX2)/4, OPG, RANKL, β-catenin, BMP-2, Runx-2, Osterix and horseradish peroxidase (HRP)-conjugated antibodies were supplied from Santa Cruz Biotechnology (Dallas, TX). Primary antibodies against osteocalcin, osteopontin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Millipore (Darmstadt, Germany). Oxygen consumption rate kit was purchased from Abcam (Cambridge, MA). 4′,6-Diamidino-2-phenylindole and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) were obtained from Invitrogen (Carlsbad, CA). Alpha minimum essential medium (α-MEM) culture media was purchased from Gibco (Grand Island, NY). Penicillin and streptomycin were purchased from American Type Culture Collection (ATCC, Manassas, VA).
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6

T Cell Purification and Gene Expression

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T cells were purified from peripheral blood using the Rosette Sep T cell purification kit (Stem Cell Technologies Inc., Vancouver, CA). Jurkat cells (clone E6-1) and 293T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). The pcDNA3.1-SRSF1-HA plasmid was a gift from Dr. James Manley (Columbia University, NY). The pGL2-zeta promoter-luciferase constructs were a gift from Dr. Barbara Rellahan [25 (link), 26 (link)] and pGL2-basic vector from Promega (Madison, WI). SRSF1 antibody was purchased from Life technologies (Carlsbad, CA), CD3ζ χλoνε 6B.10) and horseradish peroxidase (HRP)-conjugated antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO). SRSF1 specific siRNA and control siRNA were purchased from Qiagen (Valencia, CA)
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7

SDS-PAGE Analysis of rAAV Purity

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The purity of the rAAV serotype was examined by SDS-PAGE. rAAV samples with 1010 viral genomes were boiled in the 5 x oading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 200 mM DTT, 0.2% bromophenol blue, and 20% glycerol) for 5 min, and loaded on a 10% polyacrylamide gel. The samples were separated by SDS-PAGE and analyzed via sequential Western blotting as described previously [70 (link),73 (link)]. Briefly, the samples were transferred to the nitrocellulose membrane (Amersham Protran 0.45 µm Membrane; Cytiva, MA, USA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween (TBST) and incubated with the primary antibody overnight. The membrane was washed thrice in TBST and incubated with the secondary horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz, 1:3000) for one hour. Immunoreactive proteins were further detected by the ECL Western blotting substrate (Gibco, MD).
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8

Immunoblot Analysis of Signaling Pathways

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Immunoblot analyses were performed as described previously [11] . Cell lysates were prepared by adding SDS-PAGE sample solution to cells and boiled for 5 minutes. Proteins were separated by SDS-PAGE on polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore). The membrane was blocked with 2% skim milk for 1 hour, followed by incubation with the first antibody overnight at 4°C. Horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology) was used as secondary antibody. The bands were detected by ECL system (GE Healthcare). Densitometric analyses of the detected bands were performed with Image-J software.
The first antibodies used for this study are as follows; anti-phospho-STAT3 (#9131), anti-phospho-ERK1/2 (#9101), anti-ERK1/2 (#9102), anti-phospho-JAK2 (#3771), anti-JAK2 (#3230) and anti-CHOP (#2895) antibodies were purchased from Cell Signaling Technology. Anti-STAT3 (sc-7179), anti-gp130 (sc-656), anti-phospho-JAK1 (sc-16773), anti-JAK1 (sc-7228), anti-LIF receptor (LIFR) (sc-659), anti-IL-11 receptor α (IL-11Rα) (sc-993) and anti-PTP1B (sc-1718) antibodies were obtained from Santa Cruz Biotechnology. Anti-GAPDH antibody (MAB374) was purchased from Millipore, and anti-Bip/GRP78 (610978) antibody was from BD Biosciences.
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9

Sciatic Nerve Protein Analysis

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Proteins expressed in the sciatic nerve were determined by western blot analysis. Sciatic nerves were rapidly dissected from rats and used for protein analysis. All the procedures including protein extraction, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and western blotting were essentially the same as described in our previous report [8 (link)]. Briefly, cells in the sciatic nerves were lysed using triton lysis buffer, sonicated, and centrifuged to collect the supernatant containing proteins. Protein (15 μg) was used for SDS-PAGE (10%) and western blotting analyses. Anti-phospho-Erk1/2 (1 : 1250, rabbit polyclonal, Cell Signaling, Seattle, USA), anti-p38 (1 : 1250, rabbit polyclonal, Santa Cruz Biotech.), anti-TNF-α (1 : 1250, rabbit polyclonal, Sigma), and anti-actin (1 : 10,000, mouse monoclonal, MP Biomedicals, Santa Ana, USA) antibodies were used as primary antibodies, and horseradish peroxidase- (HRP-) conjugated antibody (1 : 1250; goat anti-rabbit or goat anti-mouse, Santa Cruz Biotech.) as a secondary antibody. Protein band intensity in the scanned images of X-ray film was determined by using the i-Solution software (Image & Microscope Technology, Daejeon, Korea).
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10

Western Blot Analysis of Signaling Proteins

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Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and antibiotics were purchased from Life Technologies (Rockville, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, USA). BBR was purchased from Sigma (St. Louis, USA). SR11302 was purchased from Tocris (Ellisville, USA). The secondary horseradish peroxidase (HRP)-conjugated antibody and mouse monoclonal anti–β-actin antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, USA). Rabbit monoclonal anti-FN antibodies to phospho (p)-c-Jun, total (t)-c-Jun, and c-Fos were purchased from AbCam (Cambridge, UK). The enhanced chemiluminescence (ECL) prime reagents were from Amersham (Buckinghamshire, UK).
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