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Anti rabbit igg horseradish peroxidase

Manufactured by Cell Signaling Technology
Sourced in United States

Anti-rabbit IgG-horseradish peroxidase is a conjugate of anti-rabbit immunoglobulin G (IgG) antibody and horseradish peroxidase enzyme. It is commonly used as a secondary antibody in various immunoassay techniques.

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11 protocols using anti rabbit igg horseradish peroxidase

1

HL-60 Cell Culture and Reagent Preparation

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All chemicals were obtained from commercial sources. All oligomers were purchased from Invitrogen (China). The enzymes used for reverse transcription and PCR were purchased from TaKaRa (China), and the enzymes used for plasmid construction were purchased from Fermentas (USA). The compound was synthesized by our group as described previously [21] (link) and was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 10 mM as the stock solution. HL-60 (human promyelocytic leukemia cell lines) were obtained from the American Type Culture Collection and preserved at our laboratory. HL-60 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 5% CO2 at 37 °C. The antibodies employed in the study include WT1 polyclonal antibody (sc-192, Santa Cruz, CA, USA) and anti-rabbit IgG–horseradish peroxidase (#7074, Cell Signaling, MA, USA).
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2

LPS-induced Nrf2 Activation Assay

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LPS from Eschericia coli 055:B5 was purchased from Sigma (St Louis, MO, USA). Nrf2 (H300) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), anti-rabbit IgG horse radish peroxidase (HRP)-linked antibodies, Alexa Fluor 488 F(ab’)2 anti-rabbit IgG, tatabox binding protein (TBP) and HO-1 (P249) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ABS16) antibodies were purchased from Millipore (Temecula, CA, USA). Blocking antibodies against TNF receptor 1 (TNFR1) (clone #16805) and TNFR2 (clone #22210) were purchased from R&D Systems (Minneapolis, MN, USA). Anti-CD14 (HCD14) antibody was from Biolegend, San Diego, CA, USA. H2DCFDA (C-2938) was purchased from Life Technologies (Carlsbad, CA, USA). The 4',6-diamidino-2-phenylindole (DAPI) was purchased from Biolegend (San Diego, CA, USA). The diphenyleneiodonium chloride (DPI) and wortmannin inhibitors were purchased from Sigma.
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3

Quantitative Western Blot Analysis

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Western blots were performed with whole‐cell lysates. Antibodies against tubulin and HLA‐DR (Abcam PLC, Cambridge, UK) were used with a working dilution in Can Get Signal solution I (Toyobo, Tokyo, Japan), and secondary antibodies (anti‐rabbit IgG horseradish peroxidase from Cell Signaling, Danvers, MA, USA) were used at 1 : 2000 dilutions in Can Get Signal solution II (Toyobo). Signals were observed via ECL Western blotting Detection Reagents (GE Healthcare UK Ltd., Buckinghamshire, UK). The western blot images were captured using the Amersham Imager 600 (GE Healthcare UK Ltd.). Quantification (%) was calculated according to the following formula ‘[(band intensity of HLA‐DR)/ (band intensity of Tubulin bands)] × 100’ (%) by using imagej software (NIH, Bethesda, MD, USA).
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4

Investigating Integrin-Mediated Cell Adhesion

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G-Ro (molecular weight=957.1) was obtained from Ambo Institute (Daejon, Korea). Thrombin was obtained from Chrono-Log Corporation (Havertown, PA, USA). Anti-PI3K, anti-phosphor-PI3K p85 (Tyr458), anti-Akt, anti-phosphor-Akt (Ser473), anti-rabbit IgG-horseradish peroxidase, and lysis buffer were purchased from Cell Signaling (Beverly, MA, USA). Wortmannin (PI3K inhibitor) and miltefosine (Akt inhibitor) were purchased from Cayman Chemical (Ann Arbor, MI, USA). CytoSelect 48-well cell adhesion assay kits (fibronectin-coated, colorimetric format) were purchased from Cell Biolabs (San Diego, CA, USA). Eptifibatide (αIIb/β3 inhibitor), GR 144053 (αIIb/β3 inhibitor), and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminesence solution (ECL) were purchased from General Electric Healthcare (Amersham, Buckinghamshire, UK). Fibrinogen Alexa Fluor 488 conjugate was obtained from Invitrogen Molecular Probes (Eugene, OR, USA).
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5

Ginsenoside Rg3 Regulation of Platelet Function

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We purchased 20(S)-ginsenoside Rg3 from the Ambo Institute (Daejon, Korea) and thrombin and all materials for platelet aggregation from Chrono-Log Corporation (Havertown, PA, USA). We purchased all materials for buffer solution and Rp-8-Br-cAMPS, Rp-8-Br-cGMP, p-chlorophenylthio (CPT)-cAMP, 8-Br-cGMP from Sigma (St. Louis, MO, USA) and Fura 2-acetoxymethyl (AM) from Invitrogen (Eugene, OR, USA). Thapsigargin and cAMP/cGMP enzyme immunoassay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-IP3-receptor type I, anti-phosphor-IP3-receptor type I (Ser1756), anti-extracellular signal-regulated kinase (ERK) (1/2), anti-phosphor-ERK (1/2), anti-rabbit IgG-horseradish peroxidase, and cell lysis buffer were obtained from Cell Signaling (Beverly, MA, USA). Anti-β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal to CD62P (p-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminesence solution (ECL) were purchased from General Electric Healthcare (Chalfont St. Giles, Buckinghamshire, UK).
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6

Immunoprecipitation and Detection of SLAMF9

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Native mouse SLAMF9 was immunoprecipitated from wild‐type or Slamf9−/− bone‐marrow‐derived macrophages using biotinylated anti‐SLAMF9 (9318) and Dynal MyOne Streptavidin T1 magnetic beads (Thermo Fisher). Cells were lysed in phosphate‐buffered saline, 1% Triton‐X‐100 with Roche EDTA‐Free Protease Inhibitor Cocktail and lysates cleared by centrifugation before immunoprecipitation. Immunoprecipitated proteins were deglycosylated by treatment with NEB Protein Deglycosylation Mix according to the manufacturer's instructions before sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western transfer. Western blot detection of immunoprecipitated SLAMF9 was achieved using purified anti‐mouse SLAMF9 (9318) and anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling Technologies, Danvers, MA).
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7

Western Blot Analysis of Epigenetic Markers

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Heart tissue was homogenized in RIPA Buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1% Triton X‐100, 0.5% Na deoxycholate, 0.1% SDS, 1 mmol/L EDTA, and 1X Complete Mini EDTA‐free protease inhibitor; Roche, Indianapolis, IN). For in vitro experiments, cells were scraped in RIPA buffer. Samples were sonicated using a probe sonicator. Debris was removed by centrifugation. Protein levels were quantified using a BCA assay (ThermoFisher Scientific Pierce, Rockford, IL). Ten micrograms of protein were size separated using SDS‐PAGE, transferred to nitrocellulose membranes, and subjected to standard immunoblotting procedures using antibodies targeted against G9a/Ehmt2 (C6H3), H3K9me2 (D85B4), Histone H3 (D1H2), Beta Tubulin (9F3), Actin, and mTOR (7C10) according to the manufacturer's instructions (Cell Signaling, Danvers, MA). Anti‐rabbit IgG‐horseradish peroxidase (Cell Signaling) was used as a secondary antibody. Enhanced chemiluminescent detection (Amersham GE Healthcare Life Sciences, Pittsburgh, PA) was used to detect the signal. Stripping of membranes were performed with Restore Western Blot Stripping Buffer (Thermo Scientific, Grand Island, NY). The background‐subtracted integrated intensity was quantified for each protein of interest relative to the loading control using ImageJ software (US National Institutes of Health).
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8

PKC and ERK Activation Assay

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The following primary antibodies were used: rabbit polyclonal anti PKCαp-Thr638/641, anti-PKCθ p-Thr538, anti PKCδ p-Thr505, anti PKCδ, anti ERK p-Thr202/Tyr204 and anti ERK1/2 (Cell Signaling Tech., Beverly, MA). It was also used mouse anti-β actin (Sigma-Aldrich). Secondary antibodies included: anti-rabbit IgG horseradish peroxidase (1:2000, Cell Signaling Tech) and anti-mouse IgG horseradish peroxidase (1:4000, Amersham).
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9

Evaluating Pirfenidone-Induced Signaling Pathways

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Whole cell lysates were collected in 1X CHAPS buffer (Cell Signaling), from pirfenidone-treated A549 cells as well as a human CAF line. Protein concentrations were quantified using the Bio-Rad protein assay dye. Equal amounts of protein (40 μg) were loaded into the wells of a 10 % SDS-PAGE gel and resolved at 100 V for 90 min. Proteins were then transferred to a PVDF membrane, blocked, and then probed for PARP (Cell Signaling; 9542) at 1:2000, phospho-44/42 MAPK (Erk1/2) (Cell Signaling; 4370) at 1:2000, p44/42 MAPK (Erk1/2) (Cell Signaling; 9102) at 1:2000, phospho-Akt (Cell Signaling; 9271) at 1:2000, and Akt (Cell Signaling; 4691) at 1:2000, with an overnight incubation at 4 °C. GAPDH was used at 1:2000 concentration for 30 min at room temperature (Cell Signaling; 2118S). All of the primary antibodies were incubated afterward with anti-rabbit IgG horseradish peroxidase (Cell Signaling; 7074) at 1:2000 for 30 min at room temperature.
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10

Protein Extraction and Western Blot Analysis

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Total proteins were extracted from cell lines with the mixed liquor of RIPA (radioimmunoprecipitation assay) lysis buffer (Beyotime, Shanghai, China), supplemented with 0.01% EDTA (ethylenediaminetetraacetic acid) and 10% proteinase inhibitor. The protein concentration was determined using a BCA protein assay kit (Bio-Rad). The total proteins were separated by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then transferred to polyvinylidene difluoride membranes. The membranes were blocked by 5% skimmed milk for 2 h, then incubated overnight at 4°C using the primary antibody against human COX-2 (1:1,000; Cell Signaling Technology), β-actin (1:10,000; Abcam, Cambridge, MA, USA), NF-κB1 p50 (1:1,000; Cell Signaling Technology). The membranes were further incubated using horseradish peroxidase anti-rabbit IgG (1:10,000; Cell Signaling Technology) for 1 h. The protein bands were visualized with ECL (enhanced chemiluminescence) liquid (Applygen Technologies Inc., Beijing, China) using Amersham Imager 600 equipment (GE Healthcare, Chicago, IL, USA). β-actin was utilized as the loading control.
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