Anti mouse hrp conjugated antibody

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The Anti-mouse HRP-conjugated antibody is a laboratory reagent used for the detection and quantification of mouse proteins in various immunoassays. The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that catalyzes a colorimetric reaction, allowing for the visualization and measurement of the target protein.

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Lab products found in correlation

Hrp conjugated anti rabbit antibody, by Santa Cruz Biotechnology (3 mentions) Bca protein assay reagent, by Thermo Fisher Scientific (2 mentions) Immun star westernc chemiluminescence kit, by Bio-Rad (2 mentions) Synergy 2 multi mode microplate reader, by Agilent Technologies (2 mentions) Ripa buffer, by Merck Group (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Image lab software, by Bio-Rad (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Anti β actin, by Merck Group (2 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Hrp conjugatedanti gapdh antibody, by Proteintech (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions) 12 well plate, by Corning (1 mentions) Anti flag hrp, by Cell Signaling Technology (1 mentions) Anti gapdh, by Cell Signaling Technology (1 mentions) Ecl plus, by GE Healthcare (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti h3, by Merck Group (1 mentions)

12 protocols using anti mouse hrp conjugated antibody

Western Blot Analysis of CRISPR-Edited Proteins

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For Western blots, HEK293T cells were plated on 12-well plates
(Corning) and transfected with 1.5 μg of a CIRTS expression vector
and 1.3 μg of a gRNA expression vector. After 48 h, the cells were
washed twice with ice-cold PBS and lysed in 50 μL RIPA buffer (50 mM
Tris, 150 mM NaCl, 0.5% deoxycholate, 2% SDS, pH 7.4) supplemented with
protease inhibitors. After 30 min room temperature incubation, the
concentration was measured by BCA assay (Thermo Scientific). 35 μg
protein was boiled in protein loading buffer (50 mM Tris pH 6.8, 2% SDS, 10%
glycerol, 0.05% bromphenol blue, 100 mM DTT) for 10 min at 95 °C and
loaded onto a 12% SDS PAGE gel. After stacking at 70 V, the gel was run at
120 V until the dye front reached the bottom, and the proteins were
transferred onto a PVDF membrane (Millipore) and blocked in 5% nonfat milk
in TBST. Proteins were then detected using 1:500 mouse anti-CypB antibody
(Santa Cruz), followed by 1:1000 anti-mouse HRP-conjugated antibody (Santa
Cruz). The loading control GAPDH was visualized using 1:5000 HRP-conjugated
anti-GAPDH antibody (Proteintech). Membranes were imaged on a Fluor Chem R
(Protein Simple) imager after incubation with Super Signal West Pico Plus
(Thermo Scientific).

Rauch S., He E., Srienc M., Zhou H., Zhang Z, & Dickinson B.C. (2019). Programmable RNA-guided RNA effector proteins built from human parts. Cell, 178(1), 122-134.e12.

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Western Blot Protein Detection Protocol

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Cells were lysed in RIPA Buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma). Protein concentration was measured using BCA protein assay reagent (Thermo Scientific) and BioTek Synergy 2 Multi-Mode Microplate Reader. Lysates were mixed with loading buffer and boiled for 5 min; twenty-five micrograms of protein were run in NuPage 4–12% Bis-Tris Gel polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes. Nonspecific antibody binding was blocked with TBS-T (50 mM Tris, 150 mM NaCl and 0.1% Tween-20) with 5% nonfat milk for 1 h at room temperature. The membranes were incubated with primary antibodies: anti-FLAG-HRP (1:1000, Cell Signaling 2044) in 5% milk in TBS-T for 60 min at room temperature; anti-GAPDH (1:5000, Cell Signaling, clone 14C10) in 5% milk in TBS-T for 30 min at room temperature. Membranes were then washed three times with TBS-T for 15 min total. Membranes were incubated with anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (Sigma, A 6154) or anti-mouse HRP-conjugated antibody (Santa Cruz, SC-2005) diluted 1:5000 for 30 min and washed with TBS-T three times for 15 min each. Membranes were visualized using the ImmunStar WesternC Chemiluminescence Kit (Bio-Rad) and images were captured using a ChemiDoc XRS+ System and processed using ImageLab software (Bio-Rad).

Kabadi A.M., Ousterout D.G., Hilton I.B, & Gersbach C.A. (2014). Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Research, 42(19), e147.

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Immunohistochemical and Immunoblotting Analysis of Mutant Huntingtin Aggregates

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WT and R6/2 mice were sacrificed by cervical dislocation. Brains were removed and trimmed by removing the olfactory bulbs and spinal cord. The remaining brain was processed and embedded in paraffin wax and 10 μg coronal sections were cut. Four mice/group were used and immunostaining for mHtt aggregates was carried out using a mouse anti-Htt antibody (clone EM48; 1: 150; Immunological Sciences, Cat. N. MAB-94354) as recently described (Di Pardo et al., 2018 (link)). For the immunoblotting analyses, cell lysate (30 μg) was resolved on a 10% SDS-PAGE, and entire gel, including the stacking portion, was transblotted over-night at 250 mV in 0.05% SDS and 16% methanol-containing transfer buffer (Di Pardo et al., 2018 (link)). The membrane was blocked in 5% non-fat dry milk TBST for 1 h and successively immunoblotted with anti-Htt (clone EM48) antibody (1:1,000). A monoclonal anti-mouse HRP-conjugated antibody (Santa Cruz, Cat. N. sc-2005) was used as a secondary antibody. Protein bands were visualized by ECL Plus (GE Healthcare) and quantified as described above.

Di Pardo A., Pepe G., Castaldo S., Marracino F., Capocci L., Amico E., Madonna M., Giova S., Jeong S.K., Park B.M., Park B.D, & Maglione V. (2019). Stimulation of Sphingosine Kinase 1 (SPHK1) Is Beneficial in a Huntington’s Disease Pre-clinical Model. Frontiers in Molecular Neuroscience, 12, 100.

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Western Blot Analysis of Apoptosis Markers

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Cell extracts were prepared in RIPA buffer containing 25 mM Tris/HCl pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS and 1 × Complete™ protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were heated at 95 °C for 5 min, subjected to SDS-PAGE, transferred to polyvinylidene difluoride transfer membranes, incubated with primary and secondary antibodies, and visualized using Amersham™ ECL™ Prime Western Blotting Reagent (Cytiva, Marlborough, MA, USA) and Immobilon Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. Primary antibodies used were anti-cleaved PARP, anti-cleaved caspase-3, anti-caspase-8, anti-caspase-9, anti-endonuclease G (EndoG) (1:1000; Cell Signaling Technology), anti-β-actin (1:10,000; Sigma Aldrich, St. Louis, MI, USA), and anti-H3 (1:1000; Millipore). Secondary antibodies were anti-rabbit HRP-conjugated antibody (1:10,000; Santa Cruz Biotechnology, Dallas, Texas, USA) and anti-mouse HRP-conjugated antibody (1:10,000; Santa Cruz Biotechnology).

Yun Y., Shioura M., Hitotsuyanagi Y., Yotsumoto S., Takahashi Y., Aoyagi Y., Kinoshita T., Takeya K, & Inoue H. (2021). Garcinielliptone G from Garcinia subelliptica Induces Apoptosis in Acute Leukemia Cells. Molecules, 26(9), 2422.

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Western Blot Analysis of Myogenic Markers

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Cells were lysed in RIPA Buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma). Protein concentration was measured using BCA protein assay reagent (Thermo Scientific) and BioTek Synergy 2 Multi-Mode Microplate Reader. Lysates were mixed with loading buffer and incubated at 70°C for 5 min; equal amounts of protein were run in NuPage 10% Bis-Tris Gel polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes. Nonspecific antibody binding was blocked with TBST (50 mM Tris, 150 mM NaCl and 0.1% Tween-20) with 5% nonfat milk for 1 h at room temperature. The membranes were incubated with primary antibodies (anti-MyoD (Santa Cruz, Sc-32758) in 5% bovine serum albumin (BSA) in TBST, diluted 1:250, overnight at 4°C; anti-Myogenin (Santa Cruz, Sc-12732) in 5% BSA, diluted 1:250, overnight at 4°C; anti-Beta Actin (Sigma, A2066) in 5% milk in TBST, diluted 1:5 000, for 30 min at room temperature and the membranes were washed with TBST for 15 min. Membranes were incubated with anti-rabbit HRP-conjugated antibody (Sigma, A 6154) or anti-mouse HRP-conjugated antibody (Santa Cruz, SC-2005) diluted 1:5 000 for 30 min and washed with TBST for 15 min. Membranes were visualized using the ImmunStar WesternC Chemiluminescence Kit (Bio-Rad) and images were captured using a ChemiDoc XRS+ System and processed using ImageLab software (Bio-Rad).

Manandhar D., Song L., Kabadi A., Kwon J.B., Edsall L.E., Ehrlich M., Tsumagari K., Gersbach C.A., Crawford G.E, & Gordân R. (2017). Incomplete MyoD-induced transdifferentiation is associated with chromatin remodeling deficiencies. Nucleic Acids Research, 45(20), 11684-11699.

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Protein Expression Analysis in Breast Cells

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Whole cell extracts were prepared from HMEC and MDAMB231 cells using SDS Lysis buffer supplemented with protease inhibitor (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein from whole cell extracts were separated by running on gradient polyacrylamide gels (Bio-Rad, Hercules, CA), and transferred to Amersham Hybond polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). These blots were first incubated with either a 1:2000 of anti-P63 (39739) (Active Motif, Carlsbad, CA) or anti-beta-tubulin (MAB3408) (Millipore Corp., Billerica, MA) for overnight, and followed by incubation with their corresponding secondary antibodies, anti rabbit-HRP-conjugated antibody (diluted 1:2500) (sc-2030) and anti mouse-HRP-conjugated antibody (diluted 1:4000) (sc-2031) (Santa Cruz, Dallas, Texas). Proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) and ChemiDoc XRS + Imaging System with Image Lab (Bio-Rad, Hercules, CA).

Rhie S.K., Hazelett D.J., Coetzee S.G., Yan C., Noushmehr H, & Coetzee G.A. (2014). Nucleosome positioning and histone modifications define relationships between regulatory elements and nearby gene expression in breast epithelial cells. BMC Genomics, 15(1), 331.

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Evaluation of Protein Expression Levels

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Western blot analysis was performed as described previously 18 (link). Primary antibodies used in this study p21Cip1 antibody (sc-6246; 1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-retinoblastoma (Rb) antibody (sc-271930; 1:500 dilution; Santa Cruz Biotechnology), phospo-ERK antibody (9101s; 1:500 dilution; Cell signaling technology, Danvers, MA, USA), p-MEK antibody (9121; 1:500 dilution; Cell signaling technology), Cleaved-Caspase 8 (9746s; 1:500 dilution; Cell signaling technology), Cleaved-Caspase 9 (9502S; 1:500 dilution; Cell signaling technology) and HRP-conjugated β-actin (sc47778; 1:1000 dilution; Santa Cruz Biotechnology). Secondary antibodies used in this study HRP conjugated anti-mouse antibody (sc-516102; 1:1000 dilution; Santa Cruz Biotechnology) and HRP conjugated anti-rabbit antibody (sc-2357; 1:1000 dilution; Santa Cruz Biotechnology).

Song E.S., So M.K., Park H.J., Lee H., Lee Y.H., Kuk M.U., Park J., Kwon H.W., Choi J, & Park J.T. (2023). Chemical screening identifies the anticancer properties of Polyporous tuberaster. Journal of Cancer, 14(11), 2075-2084.

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Western Blot Analysis of Caspase-3 and p53

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Proteins were isolated from the lysed cells in RIPA buffer supplemented with protease inhibitors) for 30 min at 4 °C. The amount of proteins was quantified through Bradford assay, and samples were loaded in equal amounts (50 µg) into 12% SDS-polyacrylamide gels. The proteins resolved were transferred into PVDF-transfer membranes using a Bio-Rad Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The blots were probed with the appropriate antibodies for caspase-3 (Santa Cruz Biotechnology, dilution 1:200), p53 (Santa Cruz Biotechnology, dilution 1:200), and ꞵ-Actin (Santa Cruz Biotechnology, dilution 1:200). As secondary antibody, HRP-conjugated anti-mouse antibody (Santa Cruz Biotechnology, dilution 1:50.000) was used. Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare, Chicago, Il, USA) was applied before luminography for protein detection in a ChemiDoc MP System (Bio-Rad Inc., Hercules, CA, USA).

Moreno-SanJuan S., Puentes-Pardo J.D., Casado J., Escudero-Feliu J., Khaldy H., Arnedo J., Carazo Á, & León J. (2023). Agomelatine, a Melatonin-Derived Drug, as a New Strategy for the Treatment of Colorectal Cancer. Antioxidants, 12(4), 926.

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Quantification of Apoptosis Signaling Proteins

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Cells were harvested and suspended in FLICE buffer, which contained 25 mM HEPES (pH 7.4), 1 mM EDTA, 0.1% CHAPS, 10% sucrose, protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics K.K., Tokyo, Japan). Cells were lysed by adding 1% SDS. Then, the lysates were heated for 5 min at 98 °C and analyzed by SDS-PAGE, followed by western blotting using the following antibodies: anti-human RIP1 antibody (1 : 1000, monoclonal mouse IgG clone no. 334640; R&D Systems Inc., Minneapolis, MN, USA), anti-human RIP3 antibody (1 : 1000, monoclonal rabbit IgG no. 13526; Cell Signaling Technology Japan, K.K., Tokyo, Japan), anti-p53 antibody (7F5) (1 : 1000, monoclonal rabbit IgG no. 2527; Cell Signaling Technology Japan), anti-phosphor-histone H2A.X antibody (1 : 1000, monoclonal mouse IgG clone JBW301; Millipore), anti-GAPDH antibody (1 : 2000, monoclonal mouse IgG clone 6C5 (Millipore) or 1 : 2000, monoclonal rabbit IgG clone 14C10 (Cell Signaling Technology Japan)), HRP-conjugated anti-mouse antibody (1 : 2000, monoclonal goat IgG no. C2011; Santa Cruz Biotechnology, Dallas, TX, USA) and HRP-conjugated anti-rabbit antibody (1 : 2000, monoclonal goat IgG no. 7074; Cell Signaling Technology Japan).

Ito K., Eguchi Y., Imagawa Y., Akai S., Mochizuki H, & Tsujimoto Y. (2017). MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of neuronal SH-SY5Y cells. Cell Death Discovery, 3, 17013-.

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Co-Immunoprecipitation of RNA-Binding Proteins

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Cells were grown in SC medium to logarithmic phase (OD₆₀₀ = 1.0), harvested, and lysed with glass beads in lysis buffer (50 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, 10 mM NaF, 1 mM Na₃VO₄). Immunoprecipitation was carried out using an anti-Flag antibody (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 20 μl of protein A/G beads (Santa Cruz Biotech) were added to 1 ml of freshly prepared lysate and incubated for 2 hours on a turning wheel at 4°C. Immunopellets were washed seven times in lysis buffer and recovered by boiling in 2X SDS buffer for 10 min at 100°C. For co-immunoprecipitation experiments, Dhh1 was detected using a monoclonal anti-Flag antibody (Sigma-Aldrich). Puf6 and Loc1 were detected using a monoclonal anti-PAP antibody (Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated anti-mouse antibody (Santa Cruz Biotech) was utilized as a secondary antibody.

Jung D., Seo J.S., Nam J, & Kim J. (2019). Functional association of Loc1 and Puf6 with RNA helicase Dhh1 in translational regulation of Saccharomyces cerevisiae Ste12. PLoS ONE, 14(7), e0220137.

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