Goat anti mouse igg hrp

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany, Canada, China, Poland, United Kingdom

Goat anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and visualize mouse immunoglobulin G (IgG) in various immunoassays and immunochemical techniques.

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430 protocols using goat anti mouse igg hrp

Glycoprotein Immunofluorescence Detection

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The following antibodies were used: FITC-human CD227 (Muc1) (559774, BD Biosciences), human CD227 (555925, BD Biosciences) (Muc1), Alexa-488-human podocalyxin (222328, R&D Systems), actin (sc1615, Santa Cruz), goat anti-mouse IgG-HRP (sc-2005, Santa Cruz), and mouse anti-goat IgG-HRP (sc-2354, Santa Cruz). Lectins used were: biotinylated peanut agglutinin (PNA; B-1075, Vector Laboratories), CF568 Arachis hypogaea Lectin PNA (29061, Biotium), CF640R Arachis hypogaea lectin PNA (29063, Biotium), CF633 wheat germ agglutinin (WGA; 29024, Biotium), and FITC concanavalin A (ConA; FL-1001, Vector Laboratories). Biotinylated lectins were detected using ExtrAvidin-Peroxidase (E2886, Sigma). MAPK inhibitor was U-0126 (70970, Cayman Chemical). To induce transactivator cell lines, doxycycline was used (sc-204734, Santa Cruz).

Shurer C.R., Colville M.J., Gupta V.K., Head S.E., Kai F., Lakins J.N, & Paszek M.J. (2017). Genetically Encoded Toolbox for Glycocalyx Engineering: Tunable Control of Cell Adhesion, Survival, and Cancer Cell Behaviors. ACS biomaterials science & engineering, 4(2), 388-399.

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Western Blot Analysis of Cell Signaling

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Cells were washed with PBS and lysed in 1× RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) + Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Protein quantification was performed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were prepared by adding Laemmli buffer + β-mercaptoethanol (both from BioRad) to equal volumes of protein and boiling. After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology). The membrane was washed with TBST, then incubated with an appropriate secondary: mouse anti–goat IgG–HRP (sc-2354, Santa Cruz Biotechnology), goat anti–rabbit IgG–HRP (111-036-045, Jackson ImmunoResearch), or goat anti–mouse IgG–HRP (sc-2005, Santa Cruz Biotechnology). The membrane was then washed again before addition of Clarity Western ECL Substrate (Bio-Rad). Membranes were imaged on the Odyssey Fc Imaging System (LI-COR Biosciences). Blots were quantified using Image Studio (LI‑COR Biosciences).

Prinzing B., Schreiner P., Bell M., Fan Y., Krenciute G, & Gottschalk S. (2020). MyD88/CD40 signaling retains CAR T cells in a less differentiated state. JCI Insight, 5(21), e136093.

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Western Blot Analysis of Caspase-1

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The cells were washed with PBS and were then lysed with NP40-buffer for 1 h at 4 °C on a roller shaker and were centrifuged at 12.000× g for 10 min. The protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). The samples were diluted to a concentration of 20 µg of protein in a volume of 20 µL. The samples were separated in a 10% acrylamide gel and were transferred to a nitrocellulose membrane. After blocking the membranes with 5% BSA for 1 h at room temperature, they were incubated overnight at 4 °C with the primary antibody against caspase 1 (dilution 1:400, Abcam, ab-54932). The membranes were washed 3 × 10 min with TBS-T, were incubated with the secondary antibody (goat anti-mouse IgG HRP, 1:10.000, Santa Cruz Biotechnology) for 1 h at room temperature and were washed again with TBS-0.5% Tween. The proteins were detected with a Clarity Western ECL solution. After stripping the membranes, they were stained with the anti-GAPDH monoclonal mouse antibody (1:500, Cell Signaling Technology, Danvers, MA, USA) and with the secondary antibody (goat anti-mouse IgG HRP, 1:10.000, Santa Cruz Biotechnology Dallas, TX, USA) as described above.

Brunken F., Senft T., Herbster M., Relja B., Bertrand J, & Lohmann C.H. (2023). CoNiCrMo Particles, but Not TiAlV Particles, Activate the NLRP3 Inflammasome in Periprosthetic Cells. International Journal of Molecular Sciences, 24(6), 5108.

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Plasmid Constructs for PP1, IRF7, and TLR7 Studies

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Flag-PP1 (α, β and γ) expression plasmids cloned in Flag-CMV4 were provided by Dr. Sherry Winter [53 (link)]. Flag-IRF7 expression plasmids and its mutants were described previously [54 , 55 (link)]. 2XMyc-IRF7 plasmids were generated by subcloning of 2XMyc-IRF7 fragment (by PCR amplification from pcDNA3-IRF7) into pCMV-Tag3B (Stratagene). TLR7 cDNA was cloned from human genome (the human B cell line BJAB) by PCR amplification. Deletion and point mutants were generated by site-directed mutation (Stratagene), and verified by sequencing. PE-conjugated IRF7(p477/479) antibody (clone K47–671) for flow cytometry was purchased from BD Biosciences. The same clone with higher concentration but without conjugation was customized for immunoblotting. Mouse anti-PP1 (clone E-9), mouse anti-PP1α (clone G-4), goat anti-PP1α (clone C19), mouse anti-IRF7 (clone G-8), rabbit anti-IRF7 (clone H-246), goat anti-mouse IgG-HRP, mouse anti-rabbit IgG-HRP, and mouse anti-goat IgG-HRP, were purchased from Santa Cruz. Flag (clone M2) and Myc (clone 9E10) antibodies were from Sigma and Roche, respectively. FITC-, PE-, APC-, and PerCP-eFluor710-conjugated antibodies for flow cytometry were purchased from eBioscience. Gardiquimod (designated as “Gardi” in figure legend) and HIV-1 ssRNA40 and controls were purchased from Invivogen. IFNα2 was purchased from Sigma. Tautomycin was purchased from EMD Millipore.

Wang L., Zhao J., Ren J., Hall K.H., Moorman J.P., Yao Z.Q, & Ning S. (2016). Protein phosphatase 1 abrogates IRF7-mediated type I IFN response in antiviral immunity. European journal of immunology, 46(10), 2409-2419.

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Immunoblotting and Immunohistochemistry Experiments

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Immunoblotting and immunohistochemistry were performed using goat polyclonal GAPDH (sc-48166; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal TSP1 (sc-393504; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal TSP2 (sc-12313; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal CD36 (sc-5522; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX) bovine anti-goat IgG-CFL-647 (sc-362284, Lot#B0312) and goat anti-mouse IgG-CFL-647 (sc-362257; Santa Cruz Biotechnology, Inc.). Recombinant Human Thrombospondin1 (Catalog no. 3074-TH, R&D Systems), LH (bovine LH; AFP 117438-NHPP-NIDDK), Prostaglandin F2 alpha (Catalog no. 194578; MP Biomedicals), Recombinant Human VEGF (Catalog no. 293-VE, R&D Systems) and Recombinant bFGF-basic (bovine brain derived; Catalog no. 133-F13/CF, R&D System) were procured for cell culture studies.

Paul A., Bharati J., Punetha M., Kumar S., Mallesh V.G., Chouhan V.S., Sonwane A., Bag S., Bhure S.K., Maurya V.P., Singh G., Whitworth K.M, & Sarkar M. (2019). Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis). Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 52(3).

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Immunodetection with Goat Anti-IgG HRP

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HRP goat anti-mouse IgG (RRID: AB_631736; 1:8000; sc-2055; Santa Cruz, Heidelberg, Germany).
HRP goat anti-rabbit IgG (RRID: AB_631748; 1:8000; sc-2054; Santa Cruz, Heidelberg, Germany).

Arnold S., Kortland J., Maltseva D.V., Nersisyan S.A., Samatov T.R., Lezius S., Tonevitsky A.G., Milde-Langosch K., Wicklein D., Schumacher U, & Stürken C. (2021). Fra-2 overexpression upregulates pro-metastatic cell-adhesion molecules, promotes pulmonary metastasis, and reduces survival in a spontaneous xenograft model of human breast cancer. Journal of Cancer Research and Clinical Oncology, 148(6), 1525-1542.

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Protein Expression Profiling by Western Blot

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Equal amounts of total protein were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10% gel and transferred to a polyvinylidene fluoride membrane (Roche). The membranes were blocked with 5% milk/Tris-buffered saline plus Tween 20 (TBST) and incubated with primary antibodies against PARP-1 (sc-8007), NEK2 (sc-55601), β-actin (sc-47778) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), pro-caspase-3 (#9662), β-catenin (#9562), phospho-ERK (#9101), ERK (#9102) (all from Cell Signaling, Danvers, MA, USA), and RPL17 (ab155781, Abcam, Cambridge, UK). HRP goat anti-mouse IgG, HRP goat anti-rabbit IgG, and HRP rabbit anti-goat IgG (Santa Cruz) were used as secondary antibodies. Immunoreactive bands were visualized using the LAS-3000 Imager (Fujifilm Corporation, Tokyo, Japan).

Ko M.J., Seo Y.R., Seo D., Park S.Y., Seo J.H., Jeon E.H., Kim S.W., Park K.U., Koo D.B., Kim S., Bae J.H., Song D.K., Cho C.H., Kim K.S, & Lee Y.H. (2022). RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/β-catenin Signaling Pathways. Journal of Cancer, 13(8), 2570-2583.

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Western Blot Analysis of Protein Expression

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Cells were lysed with RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). Protein concentration was quantified with BCA protein assay kit (Pierce) and samples were subjected to SDS-PAGE followed by standard Western blot procedure. Alternatively, sorted cells were directly lysed in SDS reducing sample buffer containing Triton-X100 and βME (beta (2)- Mercaptoethanol) to prepare whole cell lysates. The following antibodies were used for analysis of protein expression: anti-HSF1 (Enzo Life Sciences), p70 S6 Kinase (Cell Signaling), HSF1 phospho-Serine 303 (abcam), HSF1 phospho-Serine 326 (abcam), phospho-S6K Threonine 389 (cell signaling), β-catenin (abcam), β-actin (Sigma-Aldrich), HuR (Abcam), mTOR (Abcam), GAPDH (Santa Cruz). Secondary antibodies used were HRP-goat anti-rat IgG, HRP-goat anti-mouse IgG, HRP-goat anti-rabbit IgG (Santa Cruz).

Chou S.D., Murshid A., Eguchi T., Gong J, & Calderwood S.K. (2014). HSF1 REGULATION OF β-CATENIN IN MAMMARY CANCER CELLS THROUGH CONTROL OF HUR / ELAVL1 EXPRESSION. Oncogene, 34(17), 2178-2188.

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Immunomodulatory Effects of Eucommia ulmoides

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Eucommia ulmoides Oliver leaf was obtained from SPH Zunyi Pharmaceutical Co., Ltd. (Zunyi, China). OVA and Freund’s Adjuvant, Complete, was purchased from Sigma-Aldrich Co., LLC (St Louis, MO, USA). EDC and ADH were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Sephadex G-150 was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). The mouse macrophage (RAW264.7) cell line was obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). Phalloidin-iFluor 555 Reagent was obtained from Abcam Inc. (Abcam, Cambridge, MA, USA) and 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Solarbio (Beijing, China). HRP goat anti-mouse IgG, IgG1, IgG2a, and IgG2b were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). IL-2, IL-4, IL-6, and an IFN-γ mouse uncoated enzyme-linked immunosorbent assay (ELISA) kit were obtained from Thermo Fisher Scientific Inc. (Asheville, NC, USA).

Feng H., Yang J., Zhi H., Hu X., Yang Y., Zhang L., Liu Q., Feng Y., Wu D, & Li H. (2021). Eucommia ulmoides Leaf Polysaccharide in Conjugation with Ovalbumin Act as Delivery System Can Improve Immune Response. Pharmaceutics, 13(9), 1384.

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Western Blot Analysis of Protein Expression

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Chou S.D., Murshid A., Eguchi T., Gong J, & Calderwood S.K. (2014). HSF1 REGULATION OF β-CATENIN IN MAMMARY CANCER CELLS THROUGH CONTROL OF HUR / ELAVL1 EXPRESSION. Oncogene, 34(17), 2178-2188.

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