Goat anti rabbit igg hrp

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Goat anti-rabbit IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP) that binds to rabbit immunoglobulin G (IgG). It is commonly used in various immunoassay techniques, such as Western blotting and ELISA, to detect and quantify the presence of rabbit-derived primary antibodies.

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Goat anti-rabbit HRP (85 citations) IgG Rabbit (4 citations)

110 protocols using goat anti rabbit igg hrp

Nuclear Protein Extraction and Western Blot

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Nuclear protein was extracted and quantified using the BCA Protein Assay Kit (Life Technologies). Protein samples (10–20 µg) were electrophoresed in 4–12% Bis–Tris gels and transferred overnight to a nitrocellulose membrane at 4 °C. Primary antibodies used were anti-HDAC2 (Santa Cruz) and anti-CHD7 (#6505, Cell Signaling). Secondary antibodies were goat anti-rabbit IgG HRP (#31461, Thermo Fisher) and rabbit anti-goat IgG HRP (#31403, Thermo Fisher).

Yao H., Hannum D.F., Zhai Y., Hill S.F., Albanus R.D., Lou W., Skidmore J.M., Sanchez G., Saiakhova A., Bielas S.L., Scacheri P., Ljungman M., Parker S.C, & Martin D.M. (2020). CHD7 promotes neural progenitor differentiation in embryonic stem cells via altered chromatin accessibility and nascent gene expression. Scientific Reports, 10, 17445.

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Bacterial Protein Extraction and Western Blot

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The cells were grown at 37 °C in LB medium to OD₆₀₀ = 1 then 1 ml of culture was centrifuged for 1 min at 14000 rpm. Cell pellets were resuspended in 1/10 volume of lysis buffer containing 50 mM HEPES pH 8.0, 200 mM NaCl, 1 mM DTT, 1 mM MgCl₂, 1 mM CaCl₂, 1 mM PMSF, 25 U/ml benzonase and 0.6 mg/ml lyzozyme. Extracts were incubated for 30 min at 37 °C then 0.5% SDS were added at room temperature for 30 min. Samples were afterwards heated at 100 °C for 10 min. Samples were run on a 12.5% SDS-PAGE and transferred to nitrocellulose membrane by electroblotting. The membrane was blocked with phosphate buffer saline (PBS) solution containing 5% milk powder (w/v), for 2 h at room temperature with shaking. Then, the membrane was incubated either with anti-GFP antibody (AgroBio) used at 1/10000 dilution and the secondary antibody, a goat anti-rabbit IgG-HRP (Thermo Fisher) used at 1/2000 or with anti-YvcK antibody used at 1/2500 dilution and the secondary antibody, a goat anti-rabbit IgG-HRP (Thermo Fisher) used at 1/2000. After three washes, the Blot membrane was reading by the LAS4000 mini Imager (by chemiluminescence).

Foulquier E, & Galinier A. (2017). YvcK, a protein required for cell wall integrity and optimal carbon source utilization, binds uridine diphosphate-sugars. Scientific Reports, 7, 4139.

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Detecting MDR1 and Ubiquitin in Tumor Cells

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The tumor cells were lysed in RIPA lysis buffer containing protease inhibitors (MedChemExpress, USA) and phosphatase inhibitors (MedChemExpress, USA), and MDR1 was determined using anti-MDR1 (1:1,000, Abcam, ab170904, UK) as the primary antibody and goat anti-rabbit IgG HRP as the secondary antibody (1:5,000, eBioscience, USA). Actin served as the endogenous control.
For ubiquitin detection, an equal amount of protein (20 µg) was loaded from all samples, as assessed by Coomassie blue-stained SDS-PAGE. Anti-ubiquitin antibody (1:1,000, Sigma, #3933, USA) served as the primary antibody, and goat anti-rabbit IgG HRP (1:5,000, eBioscience, USA) served as the secondary antibody.

Huang F., Pan N., Wei Y., Zhao J., Aldarouish M., Wang X., Sun X., Wen Z., Chen Y, & Wang L. (2021). Effects of Combinatorial Ubiquitinated Protein-Based Nanovaccine and STING Agonist in Mice With Drug-Resistant and Metastatic Breast Cancer. Frontiers in Immunology, 12, 707298.

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Immunohistochemical Staining Protocol

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Primary antibodies used were: rabbit anti-DIP2B (1:2,000; Sigma HPA046133), mouse anti-α tubulin (1:5,000; Abcam ab7291), mouse anti-ace α tubulin (1:5,000; Sigma T7451), chicken anti-LacZ (1:2,000; Aves AB_2313508), mouse anti-tau1 (1:1,000; Abcam ab75714), rabbit anti-CamKII (1:500; Abcam ab52476), mouse anti-GABA (1:500; Sigma A0310), mouse anti-NeuN (1:800; Millipore MAB377), rabbit anti-GFAP (1:500; Abcam ab7260), chicken anti-GFP (1;2,000; Aves AB_2307313), mouse anti-GAPDH (1:5,000; Transgen HC301-01), rabbit anti-GST (1:2,000; CST 2625), mouse anti-His (1:2,000; Abcam ab18184).
Secondary antibodies used were: anti-Mouse IgG-HRP (1:5,000; Santa Cruz Biotechnology sc-2005), Goat-anti-Rabbit IgG-HRP (1:5,000; Invitrogen PI31460), Donkey anti-Mouse Alexa Fluor 488 (1:5,000; Invitrogen A21202), Goat anti-Chicken Alexa Fluor 488 (1:5,000, Invitrogen A11039), Donkey anti-Rabbit Alexa Fluor 546 (1:5,000, Invitrogen A10040), Donkey anti-Mouse Alexa Fluor 546 (1:5,000, Invitrogen A10036).

Xing Z.K., Zhang L.Q., Zhang Y., Sun X., Sun X.L., Yu H.L., Zheng Y.W., He Z.X, & Zhu X.J. (2020). DIP2B Interacts With α-Tubulin to Regulate Axon Outgrowth. Frontiers in Cellular Neuroscience, 14, 29.

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Western Blot Analysis of IL-17C

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Western blot was conducted as described previously.¹⁴ (link) The primary antibodies were rabbit anti-IL-17C antibody (#PA5-79474, Invitrogen, RRID: AB_2746590) and mouse anti-α-Tubulin (#66031-I-Ig, Proteintech, RRID: AB_11042766). IL-17C levels were normalized with α-Tubulin. Secondary antibody was from Invitrogen (goat anti-rabbit IgG-HRP, #UH283636; goat anti-mouse IgG-HRP, #UB278606).

Zhang F., Yin J., Liu L., Liu S., Zhang G., Kong Y., Wang Y., Wang N., Chen X, & Wang F. (2023). IL-17C neutralization protects the kidney against acute injury and chronic injury. eBioMedicine, 92, 104607.

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Evaluating LILRB Expression in Liver Cancer

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Paraffin-embedded liver cancer tissues were sectioned into 3-μm pieces. Tumor sections were incubated with rabbit polyclonal antibodies against LILRB1, LILRB2, LILRB4, and LILRB5 and rat polyclonal antibodies against LILRB3 at 4 °C for 12 h. The antibodies against LILRB1, LILRB3, LILRB4, and LILRB5 were from Abcam (product code: ab229186, ab271287, ab229747, ab121357) and the antibody against LILRB2 was from Invitrogen (product code: PA5-103913). To detect primary antibody binding, the sections were conjugated with goat anti-rabbit IgG H&L (HRP) (Abcam, Cambridge, UK), goat anti-rat IgG H&L (HRP polymer) (Abcam, Cambridge, UK), or goat anti-rabbit IgG (HRP) (Invitrogen, Carlsbad, CA, USA) at 25 °C for 2 h. Finally, the tumor sections were visualized with 3,3-diaminobenzidine and counterstained with hematoxylin. The tumor sections were observed by light microscopy. According to the literature, the H-score was used to evaluate the results [26 (link)]. In brief, the calculation method was as follows: percentage of weak staining + 2 × percentage of moderate staining + 3 × percentage of strong staining, giving a range of 0–300.

Fan J., Wang L., Chen M., Zhang J., Li J., Song F., Gu A., Yin D, & Yi Y. (2022). Analysis of the expression and prognosis for leukocyte immunoglobulin-like receptor subfamily B in human liver cancer. World Journal of Surgical Oncology, 20, 92.

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ELISA-Based Viral Protein Binding Assay

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Binding affinities of the peptides to viral protein were detected by ELISA as described previously40 (link) with some modification. Briefly, various concentrations of peptides were coated to ELISA plates and incubated with blocking buffer at 4 °C overnight. After addition of H1N1 viral HA, NA, MERS-CoV S1, or S2 (Sino Biological Inc.) and incubation at 37 °C for 1 hour, the binding affinities of the peptides to viral protein were determined by rabbit anti-His (1:2,000, Santa Cruz Biotechnology Inc, SC-8036). Goat anti-rabbit IgG-HRP was used as the secondary antibody (1:4,000, Invitrogen, 656120). Readings were obtained in an ELISA reader (Victor 1420 Multilabel Counter; PerkinElmer).

Zhao H., Zhou J., Zhang K., Chu H., Liu D., Poon V.K., Chan C.C., Leung H.C., Fai N., Lin Y.P., Zhang A.J., Jin D.Y., Yuen K.Y, & Zheng B.J. (2016). A novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses. Scientific Reports, 6, 22008.

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Western Blot Analysis of Phosphoproteins

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2-DE gels were electrotransferred onto Hybond ECL nitrocellulose membranes (GE Healthcare, UK). Membranes were blocked with 3% bovine serum albumin (BSA) in TBS containing 1% Tween-20 (TBS-T) at room temperature for 2 h before being probed with rabbit anti-phosphoserine specific polyclonal antibody (61–8100, Invitrogen Life Technologies, CA, USA, diluted 1:1000) or mouse anti-phosphothreonine specific monoclonal antibody (13–9200, Invitrogen, diluted 1:1000), incubating overnight at 4 °C. Goat anti-mouse IgG antibody (H + L) HRP (AP308P, Millipore, Temecula, CA, USA; diluted 1:80,000) and a goat anti-rabbit IgG–HRP (65–6120, Invitrogen; diluted 1:50,000) conjugates were used to localize antibody–antigen complexes. Mouse anti-phosphotyrosine-HRP monoclonal antibody (03–7720, Invitrogen; diluted 1:3000) also was used. Signal detection was performed using the Supersignal West Pico Chemiluminescent Kit (Thermo Scientific, Rockford, USA) in a luminescent image analyzer ImageQuant LAS 4000 system (GE Healthcare).

Alonso-Morales A., González-López L., Cázares-Raga F.E., Cortés-Martínez L., Torres-Monzón J.A., Gallegos-Pérez J.L., Rodríguez M.H., James A.A, & de la Cruz Hernández-Hernández F. (2015). Protein phosphorylation during Plasmodium berghei gametogenesis. Experimental parasitology, 156, 49-60.

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Immunoprecipitation and Western Blotting Protocol

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Cells were washed two times with ice-cold PBS after treatment and lysed with ice-cold lysis buffer (0.3% CHAPS, 2 mmol·L⁻¹ EDTA, 10 mmol·L⁻¹ pyrophosphate, 10 mmol·L⁻¹ glycerophosphate, 40 mmol·L⁻¹ HEPES [pH 7.4], one tablet of EDTA-free protease inhibitors (Roche, USA) per 25 mL for 15 min. Then, we acquired the supernatant by centrifugation at 12 000 × g for 10 min. The supernatant was then incubated with primary antibody overnight at 4 °C. After that, a 50% slurry of Protein A + G Sepharose was added and incubated at 4 °C for another 2 h. The immunoprecipitates were washed 3 times with ice-cold PBS. Then, 50 µL of 1x SDS sample loading buffer was added, boiled for 10 min, and analyzed by immunoblotting. The following antibodies were used: PDGFR-β (Santa Cruz, 1:50, sc-374573), TLN1 (Abcam, 1:1 000, ab108480), FAK (CST, 1:1 000, 3285), goat anti-mouse IgG HRP (Invitrogen, 1:10 000, 31430), and goat anti-rabbit IgG HRP (Invitrogen, 1:10 000, 31460).

Cui Z., Wu H., Xiao Y., Xu T., Jia J., Lin H., Lin R., Chen K., Lin Y., Li K., Wu X., Li C, & Yu B. (2022). Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation. Bone Research, 10, 58.

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Quantification of Tyrosine Hydroxylase in Striata

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Striata were isolated from both the WT and mitoNEET KO mice and homogenized in cold lysis buffer (10-fold wet weight) containing protease inhibitors and phenylmethanesulfonylfluoride (PMSF). The homogenate was centrifuged for 15 min (14000g, 4 °C), and the supernatant was removed to a new tube. Protein in the supernatant was quantified using a BCA protein assay kit (23225, Pierce, ThermoFisher Scientific, Waltham, MA). Ten micrograms of each sample was separated using a 4–12% mini-PROTEAN precast gel running at 200 V for 35 min (Bio-Rad, Hercules, CA). The protein was transferred to a poly(vinylidene fluoride) (PVDF) membrane at 100 V for 1 h. The membrane was blocked for 1 h with reconstituted 5% nonfat dry milk in 0.2% TBST (Tris-buffered saline, 0.1% Tween 20). The membrane was incubated with a primary antibody solution in TBST (rabbit anti-TH, 1:1000, Millipore, Billerica, MA) overnight at 4 °C, washed for 5 min in PBS at room temperature, and incubated with a secondary antibody in TBST (goat anti-rabbit IgG-HRP, 1:3000, Invitrogen, ThermoFisher Scientific, Waltham, MA) for 1 h at room temperature. The blot was exposed to Pierce ECL Western blotting substrate (32106, Pierce, ThermoFisher Scientific, Waltham, MA) and analyzed on a FluorChem M chemiluminescent detector (92-15312-00, ProteinSimple, San Jose, CA).

Geldenhuys W.J., Benkovic S.A., Lin L., Yonutas H.M., Crish S.D., Sullivan P.G., Darvesh A.S., Brown C.M, & Richardson J.R. (2017). MitoNEET (CISD1) Knockout Mice Show Signs of Striatal Mitochondrial Dysfunction and a Parkinson’s Disease Phenotype. ACS chemical neuroscience, 8(12), 2759-2765.

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