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Hrp conjugated anti mouse secondary antibody

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HRP conjugated anti-mouse secondary antibody is a laboratory reagent used to detect the presence of mouse primary antibodies in various immunoassay techniques, such as Western blotting and ELISA. It is composed of antibodies raised against mouse immunoglobulins and are conjugated with the enzyme horseradish peroxidase (HRP).

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Lab products found in correlation

Myecl imager, by Thermo Fisher Scientific (2 mentions) Luminata forte western hrp substrate, by Merck Group (2 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions) Ac 15, by Abcam (1 mentions) Sc 7148, by Santa Cruz Biotechnology (1 mentions) X ray film, by Fujifilm (1 mentions) Calpeptin, by Merck Group (1 mentions) Manganese 2 chloride tetrahydrate, by Merck Group (1 mentions) Horseradish peroxidase hrp conjugated anti rabbit secondary antibody, by Abcam (1 mentions) Primescript rt enzyme mix 1, by Takara Bio (1 mentions) Sybr premix ex taqtm 2 kit, by Takara Bio (1 mentions) Mouse β actin primary antibodies, by Santa Cruz Biotechnology (1 mentions) L glutamine, by Merck Group (1 mentions) Abc peroxidase staining kit, by Thermo Fisher Scientific (1 mentions) Chemiluminescence kit, by Roche (1 mentions) Slot blot apparatus, by Bio-Rad (1 mentions) Anti mouse hrp conjugated secondary antibody, by Promega (1 mentions) Anti cd81 antibody, by Santa Cruz Biotechnology (1 mentions) Ecl plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions)

11 protocols using hrp conjugated anti mouse secondary antibody

1

Western Blot Analysis of Leptin Receptor and Caspase-3

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Cellular extracts from cell lines were prepared by dissociation in lysis buffer, and protein was measured using a BCA protein assay kit. Proteins were resolved by SDS-PAGE using the laemmli buffering system with 15% polyacrylamide running gels and 5% stacking gels, and then transferred to reinforced PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 3% skim milk for 1 hour, the membranes were incubated with primary antibodies against leptin-receptor (sc-8391, 1:500, Santa Cruz,) and caspase-3 (H-227) (sc-7148, 1:1,000, Santa Cruz) for 2 hours at room temperature. After washing, the blots were incubated for 45 minutes at room temperature with a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Abcam, Cambridge, UK). After extensive washing, the antigen-antibody complexes were visualized by exposing the membranes to X-ray film (Fuji, Japan). An antibody against beta-actin antibody (AC -15, 1:10000, Abcam, Cambridge, UK) was used to verify equal loading and transfer of total proteins.
Choi E., Byeon S.J., Kim S.H., Lee H.J., Kwon H.J., Ahn H., Kim D.H, & Chang M.S. (2015). Implication of Leptin-Signaling Proteins and Epstein-Barr Virus in Gastric Carcinomas. PLoS ONE, 10(7), e0130839.
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2

Synaptic Protein Expression Analysis

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Manganese (II) chloride tetrahydrate (MnCl2·4H2O), Calpains, Calpeptin, SynaptoGreenTMC4 (FM1-43) and L-glutamine were purchased from Sigma (Saint Louis, MO, USA). PrimeScript®RT Enzyme Mix I and SYBR®Premix Ex TaqTMII kits were from TaKaRa Biotech. Co. Ltd. Mouse β-actin primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit Syntaxin 1, VAMP-2 primary antibody were purchased from Abcam Ltd. (Hong Kong), mouse SNAP-25 N-terminal monoclonal and C-terminal polyclonal antibodies were purchased from Synaptic Systems (Germany). Horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibody and HRP conjugated anti-mouse secondary antibody were purchased from Abcam. Additional chemicals of analytical grade were obtained from local chemical suppliers.
Wang C., Xu B., Ma Z., Liu C., Deng Y., Liu W, & Xu Z.F. (2017). Inhibition of Calpains Protects Mn-Induced Neurotransmitter release disorders in Synaptosomes from Mice: Involvement of SNARE Complex and Synaptic Vesicle Fusion. Scientific Reports, 7, 3701.
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3

GADD45A Immunohistochemical Staining Protocol

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Tissues were prepared for immunohistochemical staining as previously described [10 (link)]. Paraffin-embedded sections were incubated in anti-GADD45A primary antibody (1:100 dilution; Abcam, Cambridge, USA) at 4°C overnight. After washing, sections were incubated with HRP-conjugated anti-mouse secondary antibody (1:1000 dilution; Abcam) for 1 h at 37°C. The signal was enhanced using the ABC peroxidase staining kit (ThermoFisher Scientific) and staining was detected by diaminobenzidine (DAB). Sections were counterstained with hematoxylin.
Cui D., Sajan P., Shi J., Shen Y., Wang K., Deng X., Zhou L., Hu P, & Gao L. (2017). MiR-148a increases glioma cell migration and invasion by downregulating GADD45A in human gliomas with IDH1 R132H mutations. Oncotarget, 8(15), 25345-25361.
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4

Quantifying 5-Methylcytosine Levels in Genomic DNA

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5-methylcytosine (5-MeC) levels were measured in total genomic DNA isolated from whole embryos as described (Mudbhary et al., 2014 (link)). In brief, DNA was denatured using 0.4M NaOH, neutralized using cold 2M C2H3O2NH4 and 0.1 – 1 µg of genomic DNA was blotted in duplicate onto nitrocellulose membrane using a slot blot apparatus (BioRad Laboratories, Inc.), and one membrane was incubated in anti-5MeC (1:1000; Eurogentech) followed by HRP conjugated anti-mouse secondary antibody (Promega 1:2000) and visualized via chemiluminescence kit (Roche). The other membrane was stained with methylene blue or was probed with anti-double-stranded DNA primary antibody (Abcam 1:2000) followed by HRP conjugated anti-mouse secondary antibody (1:2000; Abcam) for total gDNA detection. Bands were quantified using GelAnalyzer software and 5-MeC staining was normalized to total gDNA stained with methylene blue or blotted with anti-dsDNA for each paired sample. Ratios were compared to controls to determine the degree of DNA methylation for each treatment.
Kent B., Magnani E., Walsh M.J, & Sadler K.C. (2016). UHRF1 regulation of Dnmt1 is required for pre-gastrula zebrafish development. Developmental biology, 412(1), 99-113.
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5

Western Blot Analysis of Exosomal Proteins

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Prepared CSF exosome samples were briefly sonicated, and the total protein amount was determined by the Bradford protein assay. A total of 30 μg proteins were loaded on 4–15% gradient SDS-PAGE gels and the protein mixtures were separated by size using gel electrophoresis. Separated proteins were transferred onto a PVDF membrane (0.22 μm, Bio-Rad) at constant 2.5Amp for 10 minutes using the Bio-Rad Trans-Blot Turbo Transfer System. The membrane was blocked with 3% nonfat milk in TBST (Tris buffer saline with 0.1% Tween 20) then incubated with the appropriate primary antibody overnight at 4°C. On the following day, the membrane was incubated with anti-CD81 antibody (1:200, clone B-11, Santa Cruz) and subsequently with HRP-conjugated anti-mouse secondary antibody (1:5000, abcam) diluted in TBST for 1 hour at RT. Bands were visualized by ECL Plus chemiluminescent substrate (Thermo Fisher Scientific) at the Bio-Rad ChemiDoc MP imaging system.
Yelick J., Men Y., Jin S., Seo S., Espejo-Porras F, & Yang Y. (2020). Elevated exosomal secretion of miR-124-3p from spinal neurons positively associates with disease severity in ALS. Experimental neurology, 333, 113414.
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6

Whole Cell Protein Extraction and Western Blot Analysis

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The whole cell proteins were extracted using M-PER™ Mammalian Protein Extraction Reagent (Fisher, Waltham, MA, USA). The SDS-PAGE was performed by loading 20 µg of protein to 1.0-mm NuPAGE™ 4 to 12% Bis-Tris gels, then transferred to PVDF membrane using PowerEaseTM Touch Power Supply (Fisher). The blotted membrane was detected using primary mouse anti-human antibody, horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody (Abcam, Cambridge, UK), and Luminata Forte Western HRP substrate (Millipore, Boston, MA, USA). The SDS-PAGE gel and blotted membrane were imaged with MyECL imager (Thermo Scientific, Waltham, MA, USA) and quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Original western blots data is shown in File S1.
Si Y., Zhang Y., Ngo H.G., Guan J.S., Chen K., Wang Q., Singh A.P., Xu Y., Zhou L., Yang E.S, & Liu X.(. (2021). Targeted Liposomal Chemotherapies to Treat Triple-Negative Breast Cancer. Cancers, 13(15), 3749.
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7

Western Blot Analysis of HBZY-1 Proteins

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Proteins extracted from the HBZY-1 cells were analyzed by Western blotting. Equal amounts of protein (about 50 μg) were subjected to SDS-PAGE and transferred to a PVDF membrane, then blocked in 5% skimmed milk for 2 hours at room temperature, and then incubated overnight at 4°C with the following primary antibodies according to the instructions: anti-BKCa-α, anti-BKCa-β, anti-Col IV, anti-FN, and anti-β-actin (Abcam Biotechnology, USA). The membranes were then incubated with HRP-conjugated secondary anti-mouse antibody (Abcam Biotechnology, USA). Protein bands on the membrane were visualized by ECL (electrochemiluminescence) (ThermoScientific, Rockford, IL, USA) and quantitated using QuantityOne software (Bio-Rad, Richmond, CA, USA). Image J software was used to open the strip picture to get the gray statistics of the selected area.
Wu Z., Yin W., Sun M., Si Y., Wu X, & Chen M. (2020). BKCa Mediates Dysfunction in High Glucose Induced Mesangial Cell Injury via TGF-β1/Smad2/3 Signaling Pathways. International Journal of Endocrinology, 2020, 3260728.
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8

SDS-PAGE Protein Detection Protocol

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The NuPAGE™ 4–12% Bis-Tris protein gels were used to run nonreducing SDS-PAGE. The primary mouse antihuman antibody and horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody (Abcam, Cambridge, UK) were used to detect the intracellular biomarkers and followed with treatment using Luminata Forte Western HRP substrate (Millipore, Boston, MA, USA). The blotted membrane was imaged with MyECL imager (Thermo Scientific, Waltham, MA, USA) and quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Si Y., Guan J., Xu Y., Chen K., Kim S., Zhou L., Jaskula-Sztul R, & Liu X.M. (2020). Dual-Targeted Extracellular Vesicles to Facilitate Combined Therapies for Neuroendocrine Cancer Treatment. Pharmaceutics, 12(11), 1079.
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9

Quantifying Beta-Adrenergic Receptor Levels in Lung Tissue

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Western blot was used to quantify the relative amount of β-AR in lung homogenates of animals from each treatment group that did not undergo lung mechanics as previously described [24 (link)]. After terminal anesthesia (intraperitoneal ketamine/xylazine), harvested lungs were rinsed in ice-cold PBS, snap-frozen in liquid nitrogen, and stored at −80° C. Tissue was resuspended in ice-cold commercial lysis buffer containing protease inhibitors (Sigma Aldrich) and then homogenized. Protein levels of lung homogenates were determined by BCA assay (Thermo-Scientific). Samples of 15 μg of protein were loaded and separated by 10% polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% Milk/5% BSA TBS-Tween and incubated in β-AR primary antibody (55 kDa, rabbit polyclonal, 1:1000; Abcam) overnight at 4° C then anti-rabbit HRP-conjugated secondary antibody for 1 hour at room temperature (goat polyclonal, 1:5000; Abcam). As a loading control, β-actin primary antibody (42 kDa, mouse monoclonal, 1:5000; Abcam) and anti-mouse HRP-conjugated secondary antibody (donkey polyclonal, 1:4000; Abcam) were used. β-AR band intensities were quantified and normalized to β-actin using ECL (Thermo Scientific). Relative intensities were measured using densitometry software (Image J).
Raffay T., Kc P., Reynolds J., Di Fiore J., MacFarlane P, & Martin R. (2014). Repeated beta-2 adrenergic receptor agonist therapy attenuates the response to rescue bronchodilation in a hyperoxic newborn mouse model. Neonatology, 106(2), 126-132.
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10

Quantifying SARS-CoV-2 Titers in Lungs

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For quantification of viral titers in the lungs tissues of infected animals, the middle, inferior and post-caval lobes of infected mice/hamsters were collected and homogenized in 1ml of ice-cold PBS. Lysates were then centrifuged at 10000rpm for 5min to remove cellular debris, and the cleared lysates transferred to new tubes. Lysates were then diluted in 10-fold dilutions 6 times. Quantification of infectious SARS-CoV-2 titers was then performed by plaque assays. Briefly, Vero-E6 cells were plated as confluent monolayers in 12 well dishes. Media was removed, and wells washed in 1ml of PBS. 200ul of diluted lysates was then added per well and allowed to incubate for 1 hour at 37°C. After viral adsorption, lysates were removed from the well and cells were overlaid with Minimum Essential Media supplemented with 2% FBS, 4 mM L-glutamine, 0.2% BSA, 10 mM HEPES and 0.12% NaHCO3 and 0.7% agar. 72h post infection, agar plugs were fixed in 10% formalin for 24h before being removed. Plaques were visualized by immune staining with anti-mouse SARS-CoV-2 Nucleoprotein antibodies (mAb 1C7) for 1 hour at RTP followed by anti-mouse HRP-conjugated secondary antibody (abcam) for 1 hour at RTP after 3 washes in PBS + 0.1% Tween 20. Plaques were then developed using the TrueBlue substrate (KPL-Seracare) and viral titers calculated and expressed as PFU/ml
Ho J.S., Mok B.W., Campisi L., Jordan T., Yildiz S., Parameswaran S., Wayman J.A., Gaudreault N.N., Meekins D.A., Indran S.V., Morozov I., Trujillo J.D., Fstkchyan Y.S., Rathnasinghe R., Zhu Z., Zheng S., Zhao N., White K., Ray-Jones H., Malysheva V., Thiecke M.J., Lau S.Y., Liu H., Zhang A.J., Lee A.C., Liu W.C., Jangra S., Escalera A., Aydillo T., Melo B.S., Guccione E., Sebra R., Shum E., Bakker J., Kaufman D.A., Moreira A.L., Carossino M., Balasuriya U.B., Byun M., Albrecht R.A., Schotsaert M., Garcia-Sastre A., Chanda S.K., Miraldi E.R., Jeyasekharan A.D., TenOever B.R., Spivakov M., Weirauch M.T., Heinz S., Chen H., Benner C., Richt J.A, & Marazzi I. (2021). TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation. Cell, 184(10), 2618-2632.e17.
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