Hrp conjugated anti rabbit or anti mouse antibodies

Manufactured by Jackson ImmunoResearch

Sourced in United Kingdom, United States

HRP-conjugated anti-rabbit or anti-mouse antibodies are secondary antibodies that are conjugated with the enzyme Horseradish Peroxidase (HRP). These antibodies are used to detect and visualize the presence of primary antibodies that are raised against rabbit or mouse antigens in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (3 mentions) Anti igm f ab 2 fragment, by Jackson ImmunoResearch (2 mentions) Chemidoc imaging system, by Bio-Rad (2 mentions) Chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Cell Signaling Technology (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) P erk, by Cell Signaling Technology (1 mentions) P src, by Cell Signaling Technology (1 mentions) Pstat3 ser, by Cell Signaling Technology (1 mentions) Cdc42, by Cell Signaling Technology (1 mentions) Cd40l, by R&D Systems (1 mentions) Anti β actin antibody, by Merck Group (1 mentions) Luminor enhancer solution kit, by Thermo Fisher Scientific (1 mentions) Total erk, by Cell Signaling Technology (1 mentions) Mini protean system, by Bio-Rad (1 mentions) Ab106527, by Abcam (1 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Gata3, by BD (1 mentions) Sc 47724, by Santa Cruz Biotechnology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

HRP-conjugated antibodies (35 citations) HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (16 citations) Rabbit anti-HRP (12 citations) Anti-rabbit antibodies (8 citations) Anti-rabbit or anti-mouse HRP antibodies (5 citations) Anti-mouse antibodies (5 citations) HRP anti-mouse (5 citations) Anti-rabbit HRP antibodies (4 citations) Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG antibodies (4 citations) HRP conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (3 citations)

6 protocols using hrp conjugated anti rabbit or anti mouse antibodies

Signaling Pathway Analysis in Purified B Cells

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Purified B cells were left at 37°C for 10 min in chamber buffer (PBS, 0.5% FCS, 1 g/liter d-Glucose, 2 mM MgCl2, and 0.5 mM CaCl₂) to equilibrate before stimulation. They were then stimulated for various times with 10 µg/ml anti-IgM F(ab′)₂ fragment (Jackson ImmunoResearch Laboratories), 10 µg/ml anti-κ (HB558; American Type Culture Collection), or 1 µg/ml CD40L (R&D Systems).
For Cdc42-GTP pull-down experiment, a commercial kit (Cytoskeleton Inc.) was used following the manufacturer’s instructions, specifically using 10⁷ B cells for 15 µg of beads. For immunoblotting, stimulated cells were then lysed in lysis buffer (20 mM Tris-HCL, pH 8.0,150 mM NaCl, 5 mM EDTA, Protease Inhibitor cocktail [Roche], 10 mM NaF, 1 mM Na₃VO₄, and 1% NP-40) for 15 min on ice, and samples were loaded into 4–20% (for Cdc42-GTP pull-down) or 12% (for all other applications) for electrophoresis. Proteins were detected with antibodies against pErk, pAkt (S473), pCD19, pSyk, pSrc, totalErk, pSTAT3 (Ser), and Cdc42 (all from Cell Signaling Technology) using the secondary HRP-conjugated anti–rabbit or anti–mouse antibodies (Jackson ImmunoResearch Laboratories).
Blot densitometry analysis was performed using the ImageJ (National Institutes of Health) software.

Burbage M., Keppler S.J., Gasparrini F., Martínez-Martín N., Gaya M., Feest C., Domart M.C., Brakebusch C., Collinson L., Bruckbauer A, & Batista F.D. (2015). Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity. The Journal of Experimental Medicine, 212(1), 53-72.

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OATP Protein Detection in Cell Lysates

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Whole cell lysates of Sf9 or A431 cells (10–50 µg) were separated on 7.5% Laemmli SDS-PAGE gels and transferred onto PVDF membranes. Immunoblotting was performed as described in^{36 (link)}. Membranes were incubated overnight with OATP-specific antibodies or anti-β-actin antibody (A1978, Sigma). The antibodies used for the detection of OATP1B1 and 2B1 were kind gifts from Dr. Bruno Stieger (Department of Clinical Pharmacology and Toxicology, University Hospital, 8091 Zurich, Switzerland)^{55 (link)}. The antibody raised against OATP1B3 (AMAb91231) was purchased from Atlas Antibodies (Stockholm, Sweden). Secondary antibodies used were 10,000–20,000x diluted, HRP-conjugated anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch, Suffolk, UK). Luminescence was detected using the Luminor Enhancer Solution kit by Thermo Scientific (Waltham, MA, US).

Patik I., Székely V., Német O., Szepesi Á., Kucsma N., Várady G., Szakács G., Bakos É, & Özvegy-Laczka C. (2018). Identification of novel cell-impermeant fluorescent substrates for testing the function and drug interaction of Organic Anion-Transporting Polypeptides, OATP1B1/1B3 and 2B1. Scientific Reports, 8, 2630.

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B Cell Signaling Pathway Analysis

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Purified B cells were left at 37°C for 15 min in chamber buffer (PBS, 0.5% FCS, 1g.L^-1 D-Glucose, 2mM MgCl2, 0.5mM CaCl2) to equilibrate prior to stimulation. They were then stimulated for various times with 10μg.mL anti-IgM (Fab₂)’ fragment (Jackson Immunoresearch). Stimulated cells were lysed in lysis buffer (20mM Tris-HCl, pH 8, 150mM NaCl, 5mM EDTA, Protease Inhibitor cocktail (Roche), 10mM NaF, 1mM Na₃VO₄, 1% NP40) for 30 min on ice and samples loaded into Tris Glycin gels prior to electrophoresis using the miniprotean system (BioRad). Proteins were detected with antibodies against pErk, pAkt (S473), pCD19, pSyk, pSrc, total Erk (all from Cell Signalling Technology), tubulin (Sigma Aldrich) or TC10 (Proteintech) using the secondary HRP conjugated anti-rabbit or anti-mouse antibodies (Jackson Immunoresearch). Blot densitometry analysis was performed using the Image J software.

Burbage M., Keppler S.J., Montaner B., Mattila P.K, & Batista F.D. (2017). The Small Rho GTPase TC10 Modulates B Cell Immune Responses. The Journal of Immunology Author Choice, 199(5), 1682-1695.

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Western Blot Analysis of Protein Signaling

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Total protein was extracted using RIPA lysis buffer supplemented with EDTA-free Protease Inhibitor Cocktail (#4693132001; Roche, Germany), and were boiled in SDS loading buffer. 10 μg protein was separated by 10% SDS-PAGE, followed by transferring onto 0.45 μm nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in PBST and incubated with primary antibodies against ST3GAL6 (1/500, ab106527; Abcam), GATA3 (1/500, #558686; BD, USA), EGFR (1/1000, #4267; Cell Signaling Technology (CST), USA), phospho-EGFR (Tyr1068) (1/1000, #3777; CST), AKT (pan) (1/1000, #4691; CST), phospho-AKT (Ser473) (1/1000, #4060; CST), p44/42 MAPK (ERK1/2) (1/1000, #4695; CST), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1/1000, #4370; CST), STAT3 (1/1000, #9139; CST), phospho-STAT3 (Tyr705) (1/1000, #9145; CST), or GAPDH (1/1,000, sc-47724; Santa Cruz Biotechnology, USA) at 4℃ overnight. After rinse with PBST three times, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibodies (1/5,000; Jackson ImmunoResearch, USA; Table S1), followed by the detection with the Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, USA).

Dalangood S., Zhu Z., Ma Z., Li J., Zeng Q., Yan Y., Shen B., Yan J, & Huang R. (2020). Identification of glycogene-type and validation of ST3GAL6 as a biomarker predicts clinical outcome and cancer cell invasion in urinary bladder cancer. Theranostics, 10(22), 10078-10091.

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Protein Extraction and Western Blotting

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In vitro cell lysates were collected following treatment and first washed in 1X PBS followed by rapid lysis on ice using 1X RIPA buffer (Cell Signaling Technology). Lysates were then sonicated and supernatant removed after centrifugation at 4°C. For in vivo studies, tumors were procured and 5–10 mg of tissue was homogenized in 1X RIPA on ice, followed by sonication and centrifugation in an identical manner. For western blotting of nuclear lysates, isolation was performed using Nuclear Extraction Kit (Abcam) per manufacturer’s protocol. Protein quantification was performed using Pierce^™ BCA Protein Assay Kit (Thermo Fisher) as per the manufacturer’s protocol. 12.5–40 μg of protein was loaded per lane and separated by SDS-PAGE. Membranes were then transferred to a polyvinylidene difluoride (PVDF) membrane, blocked in 5% milk solution, and incubated with indicated antibodies (Supplementary Table S1) overnight at 4°C. Secondary conjugation was performed using HRP-conjugated anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch Laboratories) followed by development with chemiluminescent substrate (Thermo Fisher) and imaged using ChemiDoc Imaging System (BioRad). Immunoblots were quantified using ImageJ (NIH, Bethesda, MD) and graphed using Prism Software (GraphPad Software Inc.).

Dosch A.R., Singh S., Dai X., Mehra S., De Castro Silva I., Bianchi A., Srinivasan S., Gao Z., Ban Y., Chen X., Banerjee S., Nagathihalli N.S., Datta J, & Merchant N.B. (2021). Targeting tumor-stromal IL-6/STAT3 signaling through IL-1 receptor inhibition in pancreatic cancer. Molecular cancer therapeutics, 20(11), 2280-2290.

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Tumor Tissue Lysis and Western Blotting

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Cell lysis and Western blotting were done as described previously (10 (link)). For in vivo studies, tumors were procured, and 5–10 mg of tissue was homogenized in 1X RIPA buffer (Cell Signaling Technology). Lysates were then sonicated, and the supernatant was removed after centrifugation at 4°C. Protein quantification was performed using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) as per the manufacturer's protocol. Protein (12.5–40 μg) was loaded and separated by SDS-PAGE. Membranes were then transferred to a polyvinylidene difluoride membrane, blocked in 5% milk solution, and incubated with primary antibodies (listed in Supplementary Table S1) overnight at 4°C. Secondary conjugation was performed using HRP-conjugated anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch Laboratories), followed by development with chemiluminescent substrate (Thermo Fisher Scientific) and imaged using ChemiDoc Imaging System (Bio-Rad). Uncropped images of blots are shown in Supplementary Figs. S1 and S2.

Mehra S., Garrido V.T., Dosch A.R., Lamichhane P., Srinivasan S., Singh S.P., Zhou Z., De Castro Silva I., Joshi C., Ban Y., Datta J., Gilboa E., Merchant N.B, & Nagathihalli N.S. (2023). Remodeling of Stromal Immune Microenvironment by Urolithin A Improves Survival with Immune Checkpoint Blockade in Pancreatic Cancer. Cancer Research Communications, 3(7), 1224-1236.

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