Goat anti mouse secondary antibody

Manufactured by Merck Group

Sourced in United States, Germany

The Goat anti-mouse secondary antibody is a laboratory reagent used to detect the presence of mouse primary antibodies in various immunoassay and immunohistochemistry applications. It is a purified antibody produced in goats that specifically binds to the constant region of mouse immunoglobulins, allowing for the identification and visualization of mouse antibodies in a sample.

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Lab products found in correlation

Goat anti rabbit secondary antibody, by Merck Group (3 mentions) Axioscope, by Zeiss (2 mentions) Horseradish peroxidase, by Merck Group (2 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions) Supersignal west pico kit, by Thermo Fisher Scientific (1 mentions) Mouse monoclonal anti ha, by Merck Group (1 mentions) Gapdh, by Abcam (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by GeneTex (1 mentions) Enhanced chemiluminescence reagent, by Cytiva (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Protein g sepharose, by Roche (1 mentions) Actin 8h10d10, by Cell Signaling Technology (1 mentions) Chemiluminescence kit, by GE Healthcare (1 mentions) Gena931, by Merck Group (1 mentions) 4 to 15 gradient sds page gel, by Bio-Rad (1 mentions) Nuclear protein extraction kit, by Thermo Fisher Scientific (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)

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29 protocols using goat anti mouse secondary antibody

Rv2004c Regulates NF-κB in THP-1 Cells

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7 × 10⁶ differentiated THP-1 cells were treated with 1 µg of Rv2004c for 24 h. Untreated cells and cells treated with LPS (100 ng/ml) served as negative and positive controls, respectively. Whole-cell protein from THP-1 cells was prepared according to the protocol described earlier (26 (link)). 100 µg of total protein was subjected to SDS PAGE and transferred onto PVDF membrane. The blots were probed with mouse antihuman p65 NF-κB antibody (Abcam, USA) followed by goat anti-mouse secondary antibody (Sigma Aldrich, USA). Blots were developed in versa doc (Bio-rad) using Super signal west Pico Kit (Thermo Fischer Scientific, USA).

Doddam S.N., Peddireddy V, & Ahmed N. (2017). Mycobacterium tuberculosis DosR Regulon Gene Rv2004c Encodes a Novel Antigen with Pro-inflammatory Functions and Potential Diagnostic Application for Detection of Latent Tuberculosis. Frontiers in Immunology, 8, 712.

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Quantifying Human Complement C3

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Mouse monoclonal antibody to human C3 (Abcam, MA), goat anti-mouse secondary antibody (Sigma, MO), purified human complement component C3 protein (Quidel, CA) were purchased.

Kim H., Meyer K., Di Bisceglie A.M, & Ray R. (2014). Inhibition of C3 Convertase Activity by Hepatitis C Virus as an Additional Lesion in the Regulation of Complement Components. PLoS ONE, 9(7), e101422.

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Immunohistochemical Visualization of HA-Tagged Dopamine Transporter

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After the behavior experiments, mice were sacrificed and perfused with 4% paraformaldehyde and the mouse brains were harvested. Areas of HA tagged DATwt expression were determined by immunohistochemical staining on floating sections. A freezing microtome was used to make 60 μm coronal brain sections through the striatal region and through the midbrain. All incubations were carried out at room temperature in PBS containing 0.3% triton X-100. The sections were first incubated in a blocking buffer containing 5% goat serum for 60 minutes and then incubated in a 1:6000 dilution of the primary antibody, a mouse monoclonal anti-HA (Sigma). The sections were washed and incubated in a 1:1000 dilution of goat-anti mouse secondary antibody (sigma) followed by an incubation in a 1:1000 dilution of peroxidase-conjugated mouse anti-peroxidase (Jackson ImmunoResearch). The sections were reacted with a chromogen using a glucose oxidase-catalyzed, nickel-intensified Diaminobenzidene (DAB) procedure (Tian et al., 2008 (link)). The sections were mounted onto slides and visualized on a CarlZeiss axioscope. The immunofluorescence procedure was similar in early steps. The secondary antibody used was a goat anti mouse Cy3 secondary antibody (1:2000) (Jackson Immunoresearch, West Grove, PA). The images were visualized using a fluorescence microscope.

Wu H., O’Neill B., Han D.D., Thirtamara-Rajamani K., Wang Y, & Gu H.H. (2014). Restoration of Cocaine Stimulation and Reward by Reintroducing Wild Type Dopamine Transporter in Adult Knock-in Mice with a Cocaine-Insensitive Dopamine Transporter. Neuropharmacology, 86, 31-37.

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Westernblot Analysis of Paraplegin

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Total cellular protein (20 μg) was resolved on a 12% Bis-Tris polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane blot was incubated overnight at 4°C with the primary mouse anti-human SPG7 monoclonal antibody (dilution 1/10,000; OriGene). Following this, the membrane was incubated with goat anti-mouse secondary antibody coupled to horseradish peroxidase (dilution 1/4,000, Sigma-Aldrich). Bands were visualized using Pierce SuperSignal West Femto Chemiluminescent Substrate on a Bio-Rad ChemiDoc XL. Paraplegin expression was normalized to GAPDH (dilution 1/10,000; Abcam) expression levels using ImageJ Version 1.52u software (Schneider et al., 2012 (link)).

Wali G., Kumar K.R., Liyanage E., Davis R.L., Mackay-Sim A, & Sue C.M. (2020). Mitochondrial Function in Hereditary Spastic Paraplegia: Deficits in SPG7 but Not SPAST Patient-Derived Stem Cells. Frontiers in Neuroscience, 14, 820.

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Flow Cytometric Analysis of Liposomal Antigen

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A FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.) equipped with a 15-mW 488-nm argon-ion laser was used to characterise viral particles and liposomes in solution. Measurement of PBS buffer alone defined the size region where noise was observed and it was established that liposomal particles gave a FSC/SSC particle distinct from their suspension buffers and that these populations could be gated to allow analysis of fluorescence. Particle-associated OVA_323-339 was assessed by producing liposomes with FITC-labelled OVA_323-339 peptides and measuring particle fluorescence by flow cytometry. Particle-associated CSP was detected by staining particles with anti-CSP antibodies (mouse mAb 2A10 –a kind gift of Dr Simon Draper, Jenner Institute, University of Oxford) followed by staining with a FITC-labelled goat anti-mouse secondary antibody (Sigma-Aldrich, UK, F2772). Antibody staining steps were performed with agitation at room temperature for one hour. Flow cytometric analysis was performed on a FACSCalibur flow cytometer (Becton Dickinson, USA) and analysed using FlowJo software (Tree Star, USA).

Hills T., Jakeman P.G., Carlisle R.C., Klenerman P., Seymour L.W, & Cawood R. (2016). A Rapid-Response Humoral Vaccine Platform Exploiting Pre-Existing Non-Cognate Populations of Anti-Vaccine or Anti-Viral CD4+ T Helper Cells to Confirm B Cell Activation. PLoS ONE, 11(11), e0166383.

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Western Blot Analysis of Tagged Ty1 Proteins

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Proteins were separated by SDS-PAGE using 14% polyacrylamide gel, and then transferred onto nitrocellulose membrane at 100 V for 70 min. ProSieve PreStained Protein Ladder Plus (Lonza) marker was used as standard. Dry milk (2%) dissolved in Tris-buffered saline (TBS, pH 7.5) was used to block the membrane for 1 h at room temperature. For the detection of tagged Ty1 proteins (Gag-PR-His₆ and PR-His₆) by Western blot, we used mouse anti-His primary antibody (460693, Invitrogen) in a 1:5000 dilution (0.24 μg/ml) diluted with TTBS (TBS complemented with Tween20) containing 0.1% dry milk. The membrane was incubated with the primary antibody for 2 h at room temperature. Then, it was washed three times with TTBS for 15 min and followed by incubation with goat anti-mouse secondary antibody (A4416, Sigma) for 1 h at room temperature. After repeated washing steps (in TTBS), the proteins were detected on the membrane by using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).

Gazda L.D., Joóné Matúz K., Nagy T., Mótyán J.A, & Tőzsér J. (2020). Biochemical characterization of Ty1 retrotransposon protease. PLoS ONE, 15(1), e0227062.

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Immunoblotting of Viral and Cellular Proteins

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Infected or transfected cells were lysed in GLB as described above, and 10 μg of protein was resolved by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore) using a Bio-Rad semidry transfer apparatus and then probed with mouse anti-ApoE antibody (Sigma or Abcam), polyclonal rabbit anti-core protein (kind gift from John McLauchlan, Centre for Virus Research, Glasgow, Scotland), rabbit polyclonal anti-AP1M1, -AP2MI, -GGA1, -GGA2, or -GGA3 (Abcam), mouse monoclonal anti-EGFP, or mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (GeneTex). Washed membranes were incubated with HRP-conjugated donkey anti-sheep, donkey anti-rabbit, or goat anti-mouse secondary antibody (Sigma) and visualized using an in-house enhanced chemiluminescence system.

Mankouri J., Walter C., Stewart H., Bentham M., Park W.S., Heo W.D., Fukuda M., Griffin S, & Harris M. (2016). Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion. Journal of Virology, 90(16), 7159-7170.

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Co-Immunoprecipitation and Western Blotting Protocol

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HEK293T cells seeded in six-well plates were transfected with 4–6 μg expression plasmids. At 20–36 h post-transfection, whole-cell extracts were prepared by using Co-IP lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid sodium salt and cocktail protease inhibitor (Roche)) and incubated with primary antibodies at 4 °C overnight, then incubated with Protein G Sepharose (Roche) at 4 °C for 4–6 h. Following triple washing in lysis buffer, analysis was conducted using SDS-PAGE followed by western blot with enhanced chemiluminescence reagent (Amersham Pharmacia, Finland). Monoclonal antibodys against HA (1:5000), FLAG (1:1000), Myc (1:7000) epitope tag and the goat anti-mouse secondary antibody (1:5000) were purchased from Sigma. Mouse monoclonal antibody against human NF-κB p65 was purchased from Cell Signaling Technology.

Wang R., Huang S., Fu X., Huang G., Yan X., Yue Z., Chen S., Li Y, & Xu A. (2017). The conserved ancient role of chordate PIAS as a multilevel repressor of the NF-κB pathway. Scientific Reports, 7, 17063.

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Western Blot Analysis of Alzheimer's Biomarkers

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For western blot analysis, 60 μg of protein was fractioned on 12% SDS-PAGE and transferred to PVDF membranes. Following the transfer, samples were blocked with 5% nonfat milk in PBS containing 10% Tween-20 for 1 h and probed with primary monoclonal antibodies against Aβ1-16 (6E10, Abcam, 1:2000 dilution), Actin (8H10D10, Cell Signaling, 1:5000), PI3K (p85, Millipore, 1:1000) and phospho-PI3K (p85/phospho-Y607, Abcam, 1:500), Akt (11E7, Cell Signaling, 1:1000) and phospho-Akt (PKBα(Ser473), Millipore, 1:500), or Bad (D24A9, 1:200) and phospho-Bad (40A9, phosphor-Ser112, both Cell Signaling, 1:1000) overnight at 4 °C. After probing with primary antibodies, the PVDF membrane was washed three times with PBST and probed with goat anti-mouse secondary antibody (Sigma, 1:5000 dilution), then detected by using a chemiluminescence kit (GE, Pittsburgh, PA, USA). Following the manufacturer’s protocol, the detection solution was prepared by mixing a 1:1 ratio of reagent A (luminol chemicals) and B (peroxide solution). The sample blots were incubated with the detection solution for 5 min and then detected using a CCD camera image system (UVP, Rockland Immunochemical Inc., Limerick, PA, USA). The blot images were analyzed using the ImageJ program.

Huang S.H., Fang S.T, & Chen Y.C. (2021). Molecular Mechanism of Vitamin K2 Protection against Amyloid-β-Induced Cytotoxicity. Biomolecules, 11(3), 423.

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SARS-CoV-2 Protein Expression Analysis

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A549 cells seeded in 6-well plates at 1 × 10⁶/well were infected with 1 mL of ΔS-VRP(G), and at 18 hpi, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and protein samples were separated on a 4 to 15% gradient SDS-PAGE gel (Bio-Rad). As controls, A549-hACE2 cells were infected with SARS-COV-2 (Wuhan-Hu-1/2019) at a multiplicity of infection (MOI) of 3 and lysed in RIPA buffer. Western blot analysis was performed following the transfer of proteins onto a nitrocellulose membrane using mouse anti-nucleoprotein or S antibody (1:1,000; Sino Biological) and goat anti-mouse secondary antibody conjugated to horseradish peroxidase (GENA931; Sigma).

Malicoat J., Manivasagam S., Zuñiga S., Sola I., McCabe D., Rong L., Perlman S., Enjuanes L, & Manicassamy B. (2022). Development of a Single-Cycle Infectious SARS-CoV-2 Virus Replicon Particle System for Use in Biosafety Level 2 Laboratories. Journal of Virology, 96(3), e01837-21.

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