Donkey anti mouse igg hrp

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Donkey anti-mouse IgG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to mouse primary antibodies in various immunoassay applications.

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20 protocols using donkey anti mouse igg hrp

Western Blot Analysis of Brain apoE

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PBS-soluble brain lysates from the sequential homogenization step were analyzed for total protein concentration with a micro BCA kit (Thermo Scientific). Thirty microgram of proteins from each sample were loaded onto a NU-PAGE 4–12% Bis-Tris 15 well gel (Thermo Fisher Scientific # NP0336BOX) and the gel was run at 150 V for 1.5 h. The proteins were subsequently dry-transferred onto a PVDF membrane using the iblot2 system (Life Technologies) and blocked with 5% milk in TBS-Tween (0.05%). The membrane was incubated with anti-apoE antibody HJ15.7 [34 (link)] (or HJ15.3) and anti-β-tubulin antibodies to probe for apoE and a loading control, respectively. Donkey-anti-mouse IgG-HRP was used as secondary antibody (Santa Cruz Biotechnology # sc-2096). All blots were developed for ~ 10 s using an enhanced chemiluminescence (ECL) Ultra kit (Lumigen TMA-6) and imaged on the SynGene Imager (BioRad) at the appropriate exposure.

Huynh T.P., Wang C., Tran A.C., Tabor G.T., Mahan T.E., Francis C.M., Finn M.B., Spellman R., Manis M., Tanzi R.E., Ulrich J.D, & Holtzman D.M. (2019). Lack of hepatic apoE does not influence early Aβ deposition: observations from a new APOE knock-in model. Molecular Neurodegeneration, 14, 37.

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Western Blot Analysis of Survivin

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Cell lysate samples suspended in sample buffer were subjected to 15% SDS‐PAGE under reducing conditions. The gel was transferred onto the Immun‐Blot PVDF membrane (Bio‐Rad Laboratories, Hercules, CA, USA). The membrane was incubated with 1% BSA in TBS containing 0.1% Tween 20 (TTBS) for 1 h at room temperature to block nonspecific protein binding, followed by overnight incubation at 4°C in TTBS containing primary antibody diluted at 1:1000 (anti‐survivin) and 1:5000 (anti‐β‐actin). After washing with TTBS, the membrane was incubated with HRP‐conjugated secondary antibodies (donkey anti‐mouse IgG‐HRP or donkey anti‐rabbit IgG‐HRP [Santa Cruz Biotechnology, Dallas, TX, USA]) diluted at 1:10 000 in TTBS for 1 h at room temperature. Target protein bands were detected by using Western Lightning Plus‐ECL (PerkinElmer, Waltham, MA, USA).

Tsuneki M., Kinjo T., Mori T., Yoshida A., Kuyama K., Ohira A., Miyagi T., Takahashi K., Kawai A., Chuman H., Yamazaki N., Masuzawa M, & Arakawa H. (2017). Survivin: A novel marker and potential therapeutic target for human angiosarcoma. Cancer Science, 108(11), 2295-2305.

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Protocol for Western Blot and Immunostaining Analysis

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HT22 cells were cultured in DMEM media supplemented with 10% FBS and 1% penicillin and streptomycin [14 (link)]. Western blots were carried out as previously reported [31 (link)] using the following primary and secondary antibodies: anti-acetylated-lysine antibody (1:1000; Cell Signaling, #9441), anti-acetyl α-tubulin-Lys40 antibody (1:10,000; 6-11B-1, Santa Cruz), anti-α-tubulin antibody (1:5000; Abcam, ab18251), anti-Tyr-Tub antibody (1:5000; Sigma T9028), anti-HDAC6 antibody (1:1000; Cell Signaling, #7558), anti-ATAT1 antibody (1:1000; Novus Biologicals, NBP1-57650), donkey anti-rabbit IgG-HRP (1:20,000; Santa Cruz) and donkey anti-mouse IgG-HRP (1:20,000; Santa Cruz). Immunostaining was performed using FITC-conjugated secondary antibodies (Jackson Immuno Research Labs) as we described previously [54 (link)]. Protein identification was conducted by Science Core Facility at Harvard University (http://proteomics.fas.harvard.edu/). Briefly, after in gel trypsin digestion, a sample was submitted for single LC-MS/MS experiment that was performed on a LTQ Orbitrap Elite (Thermo Fischer) equipped with Waters^® NanoAcquity HPLC pump (Milford, MA). Raw data were submitted for analysis in Proteome Discoverer 2.1.0.81 (Thermo Fischer) software. Assignment of MS/MS spectra was performed using the Sequest HT algorithm by searching the data against UniprotKB/Swissprot database.

You Z., Zhang S., Shen S., Yang J., Ding W., Yang L., Lim G., Doheny J.T., Tate S., Chen L, & Mao J. (2018). Cognitive Impairment in a rat model of neuropathic pain: Role of Hippocampal Microtubule Stability. Pain, 159(8), 1518-1528.

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Zebrafish Protein Expression Analysis

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10 μg protein lysate of whole zebrafish (pooled from 12 animals per group) or 10 µg of podocyte cell lysate were resolved in 10 % SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Detection of protein bands was performed using horseradish peroxidase-labelled secondary antibodies and visualized using enhanced chemi-luminescence reagents (Pierce, Rockford, IL). Primary antibodies were monoclonal mouse anti-zebrafish Vegf-A antibody (R&D systems, MAB1247, Minneapolis, MN 1:1000) and anti-GAPDH (Santa Cruz, FL-335, Dallas, Texas, 1:1000). Secondary antibodies were donkey anti-mouse IgG HRP (Santa Cruz, Dallas, Texas, 1:10.000) and donkey anti-rabbit IgG HRP (Santa Cruz, Dallas, Texas 1:10.000).

Müller-Deile J., Schröder P., Beverly-Staggs L., Hiss R., Fiedler J., Nyström J., Thum T., Haller H, & Schiffer M. (2018). Overexpression of preeclampsia induced microRNA-26a-5p leads to proteinuria in zebrafish. Scientific Reports, 8, 3621.

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Nesfatin-1 Protein Expression Analysis

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Uterine tissues of the implantation sites were quickly removed and extracted the
protein with EDTA homogenization buffer, the samples were SDS-PAGE and
transferred to the PVDF membrane. The membrane was treated in a blocking
solution and incubated with rabbit anti-rat nesfatin-1 antibody (H-003-22,
Phoenix Pharmaceuticals) / anti-mouse β-actin antibody (sc-47778, Santa Cruz
Biotechnology) followed by incubation with donkey anti-rabbit IgG-HRP (sc-2313,
Santa Cruz Biotechnology) / donkey anti-mouse IgG-HRP (sc-2096, Santa Cruz
Biotechnology), respectively. By Using the ECL Plus Western Blotting Detection
Reagents (Amersham; GE Healthcare), the membrane was detected to investigate the
expression level of nesfatin-1 protein.

Chung Y., Kim H., Seon S, & Yang H. (2017). Serum Cytokine Levels are related to Nesfatin-1/NUCB2 Expression in the Implantation Sites of Spontaneous Abortion Model of CBA/j × DBA/2 Mice. Development & Reproduction, 21(1), 35-46.

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Western Blot Analysis of Cardiac Markers

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Total proteins were extracted from EBs and heart samples using RIPA lysis buffer. Protein lysates (50 μg) were electrophoresed on 5%–10% SDS-PAGE gels, then electroblotted onto polyvinylidene fluoride membranes (Immobilon, Millipore). The membranes were blocked with 5% milk in Tris-buffered saline with Tween for 1 hr and incubated at 4°C overnight with the following primary antibodies: anti-TBX5 (Abcam, ab101227); anti-GATA4 (Abcam, ab84593); anti-GAPDH (Santa Cruz Biotechnology, SC-32233); anti-DNMT1 (Cell Signaling Technology, catalog no. 5032); and anti-DNMT3A (Cell Signaling, catalog no. 2160). The membranes were then washed and incubated with the following secondary antibodies for 2 hr at room temperature: goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) (Santa Cruz, sc-2030); donkey anti-mouse IgG-HRP (Santa Cruz, sc-2318). Membranes were finally developed using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Biotechnology) and scanned for analysis of the relative level of protein expression. The intensity of each protein band was determined by densitometry and divided by GAPDH gray value for normalization.

Jiang X.Y., Feng Y.L., Ye L.T., Li X.H., Feng J., Zhang M.Z., Shelat H.S., Wassler M., Li Y., Geng Y.J, & Yu X.Y. (2017). Inhibition of Gata4 and Tbx5 by Nicotine-Mediated DNA Methylation in Myocardial Differentiation. Stem Cell Reports, 8(2), 290-304.

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Proteomic Analysis of Extracellular Vesicles

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M-PER Mammalian Protein Extraction Reagent (Cat#78503, Thermo Fisher Scientific, Waltham, MA) together with Proteinase Inhibitor Cocktail (Cat#P8340, Sigma Aldrich, St. Louis, MO) were used to extract proteins from MDA_w, MDA_KDRab27a and MDA_KDTRAF3IP2 cells or from conditioned media of MDA_w (EXO_MDAw) or MDA_KDRab27a (EXO_MDAKDRab27a) cells. After gel electrophoresis of equal amounts of protein using 12% Precise Tris-Glycine Gels (Cat#0025267, Thermo Fisher Scientific), Laemmli Sample Buffer (Cat#161-0747, BioRad Laboratories, Hercules, CA) and BenchMark Pre-Stained Protein Ladder (Cat#10748-010, Invitrogen, Carlsbad, CA) the proteins were electroblotted and the following primary antibodies were used: GAPDH (0.0002 mg/ml; Cat#ab9485, Abcam, Cambridge, MA), Rab27a (0.01 mg/ml; Cat#sc-22756, Santa Cruz Biotechnology, Inc.), TRAF3IP2 (0.01 mg/ml; Cat#WH0010758M1-100UG, Sigma-Aldrich), CD9 (0.01 mg/ml; Cat#MA1-19002, Thermo Fisher Scientific), or MHCII (0.01 mg/ml; Cat#MA1-19143, Thermo Fisher Scientific). Goat Anti-Rabbit IgG-HRP (Cat#sc-2004, Santa Cruz Biotechnology, Inc.) or Donkey Anti-Mouse IgG-HRP (Cat#sc-2318, Santa Cruz Biotechnology, Inc.) served as secondary antibodies.

Alt E.U., Wörner P.M., Pfnür A., Ochoa J.E., Schächtele D.J., Barabadi Z., Lang L.M., Srivastav S., Burow M.E., Chandrasekar B, & Izadpanah R. (2020). Targeting TRAF3IP2, Compared to Rab27, is More Effective in Suppressing the Development and Metastasis of Breast Cancer. Scientific Reports, 10, 8834.

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Anti-Inflammatory Effects Evaluation

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The cell line—macrophage cell (RAW 264.7)—used for the measurement of cell viability was purchased from the Korean Cell Line Bank (Seoul, Korea). The reagents for cell culture, fetal bovine serum (FBS), and Dulbecco’s modified eagle medium (DMEM) were purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA). The reagents used for anti-inflammatory measurement experiments, MTT, Griess reagent, RIPA lysis and extraction buffer, LPS, protease inhibitor, phosphatase inhibitor, and nuclear and cytoplasmic extraction reagents were purchased from Sigma Aldrich Co., Ltd. The iNOS antibody, COX−2 antibody, donkey anti-mouse IgG-HRP, and mouse anti-rabbit IgG-HRP for the Western blot analysis were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). JNK antibody, p-JNK antibody, ERK1/2 antibody, p-ERK1/2 antibody, and NF-kB (p65) antibody were obtained from Cell Signaling Technology (Beverly, MA, USA).

Han D.G., Bae M.J, & An B.J. (2022). Anti-Inflammatory Activity of Velvet Bean (Mucuna pruriens) Substances in LPS−Stimulated RAW 264.7 Macrophages. Molecules, 27(24), 8797.

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Western Blot Analysis of Nesfatin-1 Protein

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Hypothalamus, pituitary, and stomach, and fat were quickly removed and extracted
the protein with EDTA homogenization buffer, the samples were SDS-PAGE and
transferred to the PVDF membrane. The membrane was treated in a blocking
solution and incubated with rabbit anti-rat nesfatin-1 antibody (H-003-22,
Phoenix Pharmaceuticals) / anti-mouse β-actin antibody (sc-47778, Santa Cruz
Biotechnology) followed by incubation with donkey anti-rabbit IgG-HRP (sc-2313,
Santa Cruz Biotechnology) / donkey anti-mouse IgG-HRP (sc-2096, Santa Cruz
Biotechnology), respectively. By using the ECL Plus Western Blotting Detection
Reagents (Amersham; GE Healthcare), the membrane was detected to investigate the
expression level of nesfatin-1 protein.

Seon S., Jeon D., Kim H., Chung Y., Choi N, & Yang H. (2017). Testosterone Regulates NUCB2 mRNA Expression in Male Mouse Hypothalamus and Pituitary Gland. Development & Reproduction, 21(1), 71-78.

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Protein Extraction and Immunoblotting of U87 and U118 Cells

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M-PER Mammalian Protein Extraction Reagent (Cat#78503, Thermo Fisher Scientific, Waltham, MA) and Proteinase Inhibitor Cocktail (Cat#P8340, Sigma-Aldrich, St. Louis, MO) were used to extract proteins from U87_TRAF3IP2KD, U87_{Control shRNA}, U118_TRAF3IP2KD, and U118_{Control shRNA} cells. After gel electrophoresis of equal amounts of protein using 12% Precise Tris–Glycine Gels (Cat#0025267, Thermo Fisher Scientific), Laemmli sample buffer (Cat#161-0747, Bio-Rad Laboratories, Hercules, CA), and BenchMark Pre-Stained Protein Ladder (Cat#10748-010, Invitrogen, Carlsbad, CA), the proteins were electroblotted and the following primary antibodies were used: GAPDH (0.0002 mg/ml; Cat#ab9485, Abcam, Cambridge, MA), TRAF3IP2 (0.01 mg/ml; Cat#WH0010758M1-100UG, Sigma-Aldrich), CD31 (0.01 mg/ml; Cat#PA5-16301, Invitrogen), IL1β (0.01 mg/ml; Cat#710331, Invitrogen), IL6 (0.01 mg/ml; Cat# MA5-23698, Invitrogen), IL8 (0.01 mg/ml; Cat# PA5-86028, Invitrogen). Goat Anti-Rabbit IgG-HRP (Cat#sc-2004, Santa Cruz Biotechnology, Inc.) or Donkey Anti-Mouse IgG-HRP (Cat#sc-2318, Santa Cruz Biotechnology, Inc.) served as secondary antibodies.

Izadpanah A., Daneshimehr F., Willingham K., Barabadi Z., Braun S.E., Dumont A., Mostany R., Chandrasekar B., Alt E.U, & Izadpanah R. (2022). Targeting TRAF3IP2 inhibits angiogenesis in glioblastoma. Frontiers in Oncology, 12, 893820.

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