Hrp anti mouse

Manufactured by Thermo Fisher Scientific

The HRP-anti-mouse is a laboratory reagent designed for use in various immunoassay techniques. It consists of a horseradish peroxidase (HRP) conjugated to an antibody that specifically binds to mouse immunoglobulins. This product can be utilized as a detection agent in procedures such as enzyme-linked immunosorbent assays (ELISAs) to identify and quantify the presence of mouse-derived proteins or antibodies in samples.

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Lab products found in correlation

Femto chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions) Supersignal west pico, by Thermo Fisher Scientific (3 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Lc2001, by Thermo Fisher Scientific (1 mentions) Ibright fl1000, by Thermo Fisher Scientific (1 mentions) Qiashredder column, by Qiagen (1 mentions) Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Dynamin 1, by Thermo Fisher Scientific (1 mentions) Complete mini proteinase inhibitor mix, by Roche (1 mentions) Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions) Luminata forte, by Merck Group (1 mentions) Bradford reagent, by Bio-Rad (1 mentions) Anti parp 1, by Santa Cruz Biotechnology (1 mentions) Anti cleaved caspase 3, by Santa Cruz Biotechnology (1 mentions) Anti acetyl p53, by Cell Signaling Technology (1 mentions) Anti sirt1, by Cell Signaling Technology (1 mentions) Anti padpr, by Santa Cruz Biotechnology (1 mentions) Anti tubulin, by Abcam (1 mentions) Anti nmnat2, by Abcam (1 mentions)

Close spelling variants of this product

Anti-rabbit or anti-mouse IgG HRPs Donkey anti-mouse HRP HRP Rat anti-Mouse IgG2a antibody Anti-mouse HRP-31430 HRP-conjugated goat anti-mouse IgG HRP)-conjugated anti-mouse/anti-rabbit IgG AntibodyGoat Anti-Rabbit or Goat Anti-Mouse IgG-HRP Anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (#31460, HRP-conjugated anti-mouse TrueBlot ULTRA antibodies Goat anti-mouse IgG1-HRP

6 protocols using hrp anti mouse

Western Blot Analysis of Fibrosis Markers

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Western blot analysis was performed to validate the release of fibrosis-related proteins and to elucidate the cell signaling pathways using α-SMA (ab7817, 1:1000), type-1 collagen (ab34710, 1:1000), total and phospho-SMAD (5678S, 8828S, 1:2000), total and phospho-JNK (sc7345, 9251S, 1:2000), total and phospho-ERK (sc514302, sc7383, 1:2000), and total and phospho-p38 (sc7149, 9211S, 1: 2000) antibodies. The treated cells were washed twice with PBS and lysed with lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 250 mmol/L NaCl2, 0.5% Triton X-100, 1 mmol/L EDTA, 1% phosphatase inhibitor, 1% protease inhibitor (Sigma, St. Louis, MO)] at 30 min (SMAD, JNK, ERK, and p38) and 48 h (α-SMA and collagen) after treatments. The supernatant was collected after centrifugation at 12,000 rpm for 15 min and quantified using a BCA protein assay kit (Thermo Fisher Scientific). Protein (30 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (LC2001, Invitrogen, MA, USA). After blocking with 5% bovine serum albumin (BSA), the membrane was incubated with primary and secondary antibodies (HRP-anti-mouse; Thermo Fisher Scientific, HRP-anti-rabbit; AbFrontier, Korea, 1:5000). The signals were detected using an imaging system (iBright FL1000, Thermo Fisher Scientific) and quantified using the gel analysis plugin in ImageJ.⁴³ (link)

Lee Y., Heo S.Y., Lee H.S., Oh S.J., Kim H., Lim S., Shin H., Jung W.K, & Kang H.W. (2022). Combinatorial prophylactic effect of phlorotannins with photobiomodulation against tracheal stenosis. iScience, 25(11), 105405.

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Western Blot Protein Detection

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Cell lysates were subjected to SDS-PAGE electrophoresis using 8% gels. For experiments where multiple antibodies are probed, samples in 6x LSB were prepared in bulk. Twenty micrograms of sample from the same batch was run in multiple gels at the same time to ensure consistency. Samples were then transferred to PVDF membranes. Following blocking with 5% milk, membranes were cut horizontally to probe multiple proteins of different molecular weights on the same blot. A list of antibodies used can be found in Excel S1. Secondary HRP-anti-rabbit and HRP-anti-mouse were obtained from ThermoFisher Scientific. SuperSignal West Pico and Femto Chemiluminescent Substrates (Thermo) were used to visualize blots.

Kurimchak A.M., Shelton C., Herrera-Montávez C., Duncan K.E., Chernoff J, & Duncan J.S. (2019). Intrinsic Resistance to MEK Inhibition Through BET Protein Mediated Kinome Reprogramming in NF1-deficient Ovarian Cancer. Molecular cancer research : MCR, 17(8), 1721-1734.

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Western Blotting Protocol for Phosphoproteins

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Samples were harvested in MIB lysis buffer, subjected to SDS-PAGE chromatography and transferred to PVDF membranes before western blotting with primary antibodies. For pMYPT and pMLC2 blots, cells were harvested in a buffer containing 1% (w/v) SDS, 50 mM Tris pH 7.5 supplemented with protease and phosphatase inhibitors buffer and were passed through QIAshredder columns (Qiagen, 79654) (21 (link)). For the list of primary antibodies used, see (Data file S10). Secondary HRP-anti-rabbit and HRP-anti-mouse were obtained from ThermoFisher Scientific. SuperSignal West Pico and Femto Chemiluminescent Substrates (Thermo Scientific) were used to visualize blots. Western blot images were quantified using the Analyze>Gels function in Image J open source software (National Institutes of Health). Optical density values were normalized by sum (fig. S2, G–J) or by a fixed point (Fig. 4E, Fig. 5B and Fig. 7F) (45 (link)). Phosphorylated proteins were normalized to loading control, then normalized to total abundance of the respective protein.

Kurimchak A.M., Herrera-Montavez C., Brown J., Johnson K.J., Sodi V., Srivastava N., Kumar V., Deihimi S., O’Brien S., Peri S., Mantia-Smaldone G.M., Jain A., Winters R.M., Cai K.Q., Chernoff J., Connolly D.C, & Duncan J.S. (2020). Functional proteomics interrogation of the kinome identifies MRCKA as a therapeutic target in high-grade serous ovarian carcinoma. Science signaling, 13(619), eaax8238.

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Quantitative Western Blot Analysis

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Samples were harvested in lysis buffer (50 mM HEPES (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 2.5 mM sodium orthovanadate, 1X protease inhibitor cocktail (Roche), and 1% each of phosphatase inhibitor cocktails 2 and 3 (Sigma)). Particulate was removed by centrifugation of lysates at 21,000 rpm for 15 minutes at 4°C. Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) chromatography and transferred to PVDF membranes before western blotting with primary antibodies (table S1). Secondary HRP-anti-rabbit and HRP-anti-mouse were obtained from ThermoFisher Scientific. SuperSignal West Pico and Femto Chemiluminescent Substrates (Thermo) were used to visualize blots. Western blot images were quantified using the Analyze>Gels function in Image J open-source software (National Institutes of Health). Optical density values for total protein levels were normalized by GAPDH. DC50 (the concentration where 50% of protein has been degraded) plots and values were calculated using PRISM software. Samples were run in biological triplicates (N=3) and Student’s t-tests were performed for statistical analyses and P values ≤ 0.05 were considered significant.

Kurimchak A.M., Herrera-Montávez C., Montserrat-Sangrà S., Araiza-Olivera D., Hu J., Neumann-Domer R., Kuruvilla M., Bellacosa A., Testa J.R., Jin J, & Duncan J.S. (2022). The drug efflux pump MDR1 promotes intrinsic and acquired resistance to PROTACs in cancer cells. Science signaling, 15(749), eabn2707.

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Western Blot Analysis of Protein Extracts

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Protein extracts were made in IP lysis buffer (10 mM HEPES pH 7.5, 137 mM NaCl, 0.4% NP-40, 10% glycerol) with Complete-mini proteinase inhibitor mix (Roche) added fresh. Extracts were quantified using the Bradford reagent (Bio-Rad). Extracts (50 μg protein) were diluted in Laemmli buffer, incubated at 95°C for 5 minutes, resolved by SDS-PAGE and transferred to nitrocellulose membrane. All membrane blotting steps were carried out in TBS plus Tween (TBST). Blots were blocked in 5% milk, then incubated at RT with primary antibody (for specific dilutions see below) for 4 hours, HRP-conjugated secondary antibody (1:15000) for 1h and visualized with Luminata Forte (Millipore). Membranes were incubated with Restore Western blot stripping buffer (Thermo Scientific) at room temperature for 10 minutes while shaking to remove antibodies for subsequent hybridization. Primary antibodies used were dynamin-1 (1:3000; Thermo Scientific, PA1-660), actin (1:1000; Abcam, ab3280). Secondary antibodies used were HRP anti-mouse (Thermo Scientific), HRP anti-rabbit (BioRad).

Asinof S.K., Sukoff Rizzo S.J., Buckley A.R., Beyer B.J., Letts V.A., Frankel W.N, & Boumil R.M. (2015). Independent Neuronal Origin of Seizures and Behavioral Comorbidities in an Animal Model of a Severe Childhood Genetic Epileptic Encephalopathy. PLoS Genetics, 11(6), e1005347.

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Comprehensive Antibody Protocol for Fly Research

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The following commercially available antibodies were used: anti-Repo (1:250, DSHB, 8D12), anti-Caspase-3 (1:250 for Immunocytochemistry of fly brain, 1:1000 for Western blot analysis, Cell Signaling Technology, 9665), anti-Cleaved Caspase-3 (1:1000, Santa Cruz, 9661), anti-H2AvD (1:50, Rockland, 600-401-914), anti-p53(E-5) (1:50, Santa Cruz, sc-74573), p53(DO-1) (1:1000, Santa Cruz, sc-126), anti-Drosophila Nmnat (1:3000), anti-NMNAT1 (1:1000, Abcam, ab45548), anti-NMNAT1 (1:1000, Santa Cruz, 271557), anti-NMNAT2 (1:500, Abcam, ab56980), anti-PARP1 (1:1000, Santa Cruz, sc-8007), anti-pADPr (1:1000, Santa Cruz, sc-56198), anti-SIRT1 (1:1000, Cell Signaling Technology, 2492), anti-acetyl-p53 (1:1000, Cell Signaling Technology, 2525), anti-β-actin (1:10,000, Sigma-Aldrich, A1978), and anti-tubulin (1:300, Abcam, ab15246). The secondary antibodies conjugated to Alexa 488/546/647 (1:250, Invitrogen), or near-infrared (IR) dye 700/800 (1:5000, LI-COR Biosciences). HRP-anti-mouse and HRP-anti-rabbit (1:5000, Thermo Fisher Scientific).

Liu J., Tao X., Zhu Y., Li C., Ruan K., Diaz-Perez Z., Rai P., Wang H, & Zhai R.G. (2021). NMNAT promotes glioma growth through regulating post-translational modifications of P53 to inhibit apoptosis. eLife, 10, e70046.

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