Anti rabbit hrp conjugated antibody

Manufactured by Promega

Sourced in United States

The Anti-rabbit HRP conjugated antibody is a secondary antibody that binds to rabbit primary antibodies. The horseradish peroxidase (HRP) enzyme is conjugated to the antibody, allowing for colorimetric or chemiluminescent detection of target proteins in various immunoassay applications.

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8 protocols using anti rabbit hrp conjugated antibody

Western Blot Analysis of EphA2 Expression and Phosphorylation

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Twenty four hours following transfection, the cells were lysed in lysis buffer (25mM TrisHCl, 0.5% TritonX-100, 20mM NaCl, 2mM EDTA, 2mM Na₃VO₄, and Roche Applied Science protease and phosphatase inhibitor). The lysed samples were collected and centrifuged at 14,000g for 15 minutes at 4°C. The lysates were collected and stored at −20°C. The total protein concentrations in the lysates were measured with the BCA protein assay kit (Bio-Rad, CA). The lysates were loaded into 3–8% NuPAGE^HNovex^HTris-Acetate mini gels (Invitrogen, CA). The proteins were transferred onto a nitrocellulose membrane, and blocked using 5% non-fat milk in TBST. Total EphA2 expression, S897 phosphorylation and Y772 phosphorylation were detected using anti-EphA2 antibodies (R&D systems, MN), anti-phospho-S897 antibodies (Cell Signaling, MA), and anti-phospho-Y772 antibodies (Cell Signaling, MA), respectively. Donkey anti-goat HRP conjugated antibodies (Promega, WI) and anti-rabbit HRP conjugated antibodies (Promega, WI) were used as secondary antibodies. The membranes were incubated with Amersham ECL Plus^TM Western Blotting Detection Reagent (GE HealthCare Life Sciences, PA) for 2 minutes and then exposed for 1 to 60 seconds in a Chemidoc molecular imager (Bio-Rad, CA) to detect the chemiluminescent bands.

Singh D.R., Ahmed F., Paul M.D., Gedam M., Pasquale E.B, & Hristova K. (2016). The SAM domain inhibits EphA2 interactions in the plasma membrane. Biochimica et biophysica acta, 1864(1), 31-38.

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EphA2 Phosphorylation Quantification

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Twenty four hours following transfection, the cells were incubated with medium containing 6 μM of YSA. The cells were placed in an incubator for 10 minutes and then treated with lysis buffer (25mM Tris-Cl, 0.5% TritonX-100, 20mM NaCl, 2mM EDTA, phosphate and protease inhibitors (Roche Applied Science)). The lysed samples were centrifuged at 14,000 g for 15 minutes at 4°C and stored at −20°C. The amounts of total protein in the lysates were measured with the BCA protein assay kit (Bio-Rad, CA). The lysates were loaded into 3–8% NuPAGE^HNovex^HTris-Acetate mini gels (Invitrogen, CA). The proteins were transferred onto a nitrocellulose membrane, and blocked using 5% non-fat milk in 1×TBST. Total EphA2 expression, Ser897 phosphorylation and Tyr772 phosphorylation were probed using anti-EphA2 antibodies (Cell Signalling, MA), anti-phospho-Ser897 antibodies (Cell Signaling, MA), and anti-phospho-Tyr772 antibodies (Cell Signaling, MA), respectively. Anti-rabbit HRP conjugated antibodies (Promega) were used as secondary antibodies to visualize the EphA2 expression and phosphorylation bands. The membranes were incubated with Amersham ECL Plus™ Western Blotting Detection Reagent (GE HealthCare Life Sciences, PA) for 2 minutes and then exposed for 1 to 60 seconds in a Chemidoc molecular imager (Bio-Rad, CA) to detect the chemiluminescent bands.

Singh D.R., Pasquale E.B, & Hristova K. (2016). A Small Peptide Promotes EphA2 Kinase-Dependent Signaling by Stabilizing EphA2 Dimers. Biochimica et biophysica acta, 1860(9), 1922-1928.

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Protein Expression Analysis by Western Blot

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Protein extraction and western blot analysis were performed as previously described (Serrano et al., 2012 (link); Vida et al., 2013 (link)). The samples (50 µg of total proteins each) were resolved on 4-15% Ready Gel Precast Gels (Bio-Rad Laboratories, Inc.) and subsequently blotted onto nitrocellulose membranes (Bio-Rad). Specific proteins were detected after incubation in TBS-T containing 2% BSA and the corresponding primary antibodies: rabbit anti-ACCα/β, anti-FAS, anti-SCD1, anti-AMPKα, anti-phospho-AMPKα (Thr172), anti-STAT3, anti-phospho-STAT3 (Tyr705) and anti-actin antibodies (Cell Signaling Technology Inc., MA, USA). Rabbit anti-CPT1a and anti-adaptin γ antibodies were purchased from Abcam (Cambridge, UK). An anti-rabbit HRP-conjugated antibody was used as secondary antibody (Promega, Madison, WI, USA). The specific protein bands were revealed using the enhanced chemiluminescence detection system (Santa Cruz, Biotechnology Inc., CA, USA), according to the manufacturer's instructions, and the images were visualized using an Autochemi-UVP Bioimaging System. The bands were quantified through densitometric analysis using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij, 1997-2012). The levels of specific proteins were normalized to actin or adaptin levels.

Vida M., Gavito A.L., Pavón F.J., Bautista D., Serrano A., Suarez J., Arrabal S., Decara J., Romero-Cuevas M., Rodríguez de Fonseca F, & Baixeras E. (2015). Chronic administration of recombinant IL-6 upregulates lipogenic enzyme expression and aggravates high-fat-diet-induced steatosis in IL-6-deficient mice. Disease Models & Mechanisms, 8(7), 721-731.

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Quantification of EphA2 Phosphorylation

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Cells were transfected with plasmids using Lipofectamine 3000, grown for about 12 hours, serum-starved for ~12 hours and treated with FBS for 15 min, in some cases in the presence of 5 μg/ml (200 nM) m-ephrinA1. Cell lysates were collected with lysis buffer (25 mM Tris-Cl, 0.5% TritonX-100, 20 mM NaCl, 2 mM EDTA and phosphatase and protease inhibitors (Roche Applied Science)). The lysates were centrifuged at 14,000 g for 15 minutes at 4°C and stored at −20°C. BCA assays (Bio-Rad) were used to measure the protein concentrations of the samples. Lysates mixed with LDS sample buffer and DTT were run on 3–8% NuPAGE^HNovex^HTris-Acetate mini gels (Invitrogen, CA) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in 1× TBST. EphA2 expression was quantified using anti-EphA2 antibodies (Cell Signalling, MA). S897 and Y772 phosphorylation levels were quantified using anti-phospho-Ser897 and anti-phospho-Tyr772 antibodies (Cell Signaling, MA) followed by an anti-rabbit HRP conjugated antibody (Promega, WI). Nitrocellulose membranes were incubated for 2 minutes with Amersham ECL Plus Western Blotting Detection Reagent (GE Health Care Life Sciences, PA) and exposed from 1 to 60 seconds to capture images with the ChemiDoc imaging system (Bio-Rad, CA).

Singh D.R., Kanvinde P., King C., Pasquale E.B, & Hristova K. (2018). The EphA2 receptor is activated through induction of distinct, ligand-dependent oligomeric structures. Communications Biology, 1, 15.

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Quantifying EphA2 Phosphorylation in Cells

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Cells were transfected with plasmids using Lipofectamine 3000, grown for about 12 h, serum-starved for ~12 h and treated with FBS for 15 min, in some cases in the presence of 5 μg/ml (200 nM) m-ephrinA1. Cell lysates were collected with lysis buffer (25 mM Tris-Cl, 0.5% TritonX-100, 20 mM NaCl, 2 mM EDTA and phosphatase and protease inhibitors (Roche Applied Science)). The lysates were centrifuged at 14,000 × g for 15 min at 4 °C and stored at −20 °C. BCA assays (Bio-Rad) were used to measure the protein concentrations of the samples. Lysates mixed with LDS sample buffer and reducing buffer were run on 3–8% NuPAGE^HNovex^HTris-Acetate mini gels (Invitrogen, CA) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in 1 × TBST. EphA2 expression was quantified using anti-EphA2 antibodies (Cell Signalling, MA). S897 and Y772 phosphorylation levels were quantified using anti-phospho-Ser897 and anti-phospho-Tyr772 antibodies (Cell Signaling, MA) followed by an anti-rabbit HRP conjugated antibody (Promega, WI). Nitrocellulose membranes were incubated for 2 min with Amersham ECL Plus Western Blotting Detection Reagent (GE Health Care Life Sciences, PA) and exposed from 1 to 60 s to capture images with the ChemiDoc imaging system (Bio-Rad, CA). Uncropped images of all Western blots presented in the study are shown in Supplementary Figures 13-18.

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Trk-A Protein Crosslinking Analysis

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HEK293T cells were transfected with 2ug of Trk-eYFP DNA. A membrane impermeable crosslinker, BS³ (bis(sulfosuccinimidyl) suberate; ThermoFisher Scientific, 21580), was used in the crosslinking experiments. Twenty four hours after transfection with Trk-A-YFP DNA, cells were incubated with 2 mM BS³ for 1 hour at room temperature. The reaction was quenched with 20 mM Tris-HCl for 15 min. The cells were rinsed with ice-cold 1X PBS before lysing. In some cases, cells were incubated with the neurotrophin hß-NGF (5ug/mL) for 10–15 min before adding the crosslinker. The lysates were subjected to SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. Trk-A-eYFP was probed using anti-GFP antibody (Cell Signaling Technologies, 2555S) as the primary antibody, followed by anti-rabbit HRP conjugated antibody (Promega, W4011). The membrane was incubated with Amersham ECL Plus ™ (GE HealthCare Life Sciences, RPN2106) for 2 min and exposed to detect the chemiluminescent signal in a Chemidoc imaging system (Bio-Rad).

Ahmed F, & Hristova K. (2018). Dimerization of the Trk receptors in the plasma membrane: effects of their cognate ligands. The Biochemical journal, 475(22), 3669-3685.

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Germacrone-Induced Autophagy Signaling

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Germacrone (purity >98%) was bought from Shanghai YuanYe Biotechnology Co. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was bought from Bio Basic. Antibody against GAPDH was bought from Merck Millipore (Darmstadt, Germany). Antibodies against LC3B, mTOR, p-mTOR, Akt, and p-Akt were bought from Cell Signaling Technology (Danvers, MA). HRP-conjugated anti-rabbit antibody was bought from Promega (Madison, USA). An enhanced chemiluminescence (ECL) kit was bought from Biological Industries (Kibbutz Beit Haemek, Israel). Chloroquine and 3-methyladenine were purchased from Sigma Aldrich (St. Louis, MO). Goat anti-rabbit IgG H&L was bought from Abcam (Cambridge, United Kingdom).

Yu Z., Xu J., Shao M, & Zou J. (2020). Germacrone Induces Apoptosis as Well as Protective Autophagy in Human Prostate Cancer Cells. Cancer Management and Research, 12, 4009-4016.

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Western Blot Analysis of Protein Expression

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To check for protein expression, equal volumes of protein lysate were loaded on an 8% sodium dodecyl sulfate–polyacrylamide gel, and electrophoresis was performed. Separated proteins were transferred onto a nitrocellulose membrane (Amersham Protran 0.45, GE Healthcare, Chicago, IL, USA). The membrane was blocked with 5% non-fat milk in TBS-T buffer (Tris-buffered saline supplemented with 0.1% Tween 20) at room temperature for one hour. The blocked membrane was probed with primary antibodies targeting p-STAT1 (#9167, Cell Signaling Technology, Danvers, MA, USA, 1:1000) and PD-L1 (#13684, Cell Signaling Technology, Danvers, MA, USA, 1:1000) at 4 °C overnight. GAPDH (#2118, Cell Signaling Technology, Danvers, MA, USA, 1:1000) was used as a protein loading control. After washing, the membrane was incubated with secondary HRP-conjugated anti-rabbit antibody (Promega, Madison, WI, USA). Proteins were detected by applying an ECL Western blotting detection reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Li X., Pan K., Vieth M., Gerhard M., Li W, & Mejías-Luque R. (2022). JAK-STAT1 Signaling Pathway Is an Early Response to Helicobacter pylori Infection and Contributes to Immune Escape and Gastric Carcinogenesis. International Journal of Molecular Sciences, 23(8), 4147.

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