Anti mouse igg alkaline phosphatase secondary antibody

Manufactured by Merck Group

Sourced in United States

Anti-mouse IgG alkaline phosphatase is a secondary antibody that binds to mouse primary antibodies. It is used in various immunoassays and immunodetection techniques to amplify and visualize the signal from the primary antibody.

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Lab products found in correlation

5 bromo 4 chloro 3 indolyl phosphate nitro blue tetrazolium substrate, by Merck Group (1 mentions) Monoclonal anti polyhistidine antibody, by Merck Group (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Slot blot apparatus, by Bio-Rad (1 mentions) Nitro blue tetrazolium chloride nbt, by Merck Group (1 mentions) 5 bromo 4 chloro 3 indolyl phosphate bcip, by Thermo Fisher Scientific (1 mentions) Bcip nbt liquid substrate, by Merck Group (1 mentions)

3 protocols using anti mouse igg alkaline phosphatase secondary antibody

Western Blot Analysis of Polyhistidine-Tagged Proteins

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The purified protein was run in SDS-PAGE (12 %), followed by blotting onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 3 % BSA in 1X TBST buffer (pH 7.6). The membrane was washed three times with 1X TBST buffer. Then the membrane was incubated with monoclonal anti-polyhistidine antibody (Sigma, St. Louis, MO, USA) at 4 °C for 12 h, followed by thorough washing with 1X TBST buffer. After this, anti-mouse IgG alkaline phosphatase (secondary antibody) (Sigma, St. Louis, MO, USA) was introduced, and incubated at room temperature for 2 h. After washing with 1X TBST buffer, the membrane protein was visualized by 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium) substrate (Sigma, St. Louis, MO, USA) at room temperature [37 , 69 (link)].

Patel S.N., Kaushal G, & Singh S.P. (2021). d-Allulose 3-epimerase of Bacillus sp. origin manifests profuse heat‐stability and noteworthy potential of d-fructose epimerization. Microbial Cell Factories, 20, 60.

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SNO Quantification in AD Mice Brains

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Brain tissues from WT (N = 4) and AD (N = 4) mice were homogenized with the presence of NEM and incubated with 5% SDS for 0.5 h at room temperature. BCA assay was used to determine protein concentrations. An aliquot of 250 μL PBS containing 0.5 μg protein was loaded onto a nitrocellulose membrane with a slot blot apparatus (Bio-Rad). The membranes were blocked with 3% (w/v) BSA solution and 5 mM NEM overnight at 4°C and incubated with a 1:2500 dilution of anti-SNO antibody produced by mouse (Sigma) for 2 hours. After rinsing the membrane, anti-mouse IgG alkaline phosphatase secondary antibody (Sigma) was added with the dilution factor 1:5000 and incubated with the membrane for 1 hour. The membrane was washed in wash blot (PBS with 0.1% Tween 20) and developed using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Thermo Fisher)/nitro blue tetrazolium chloride (NBT) (Sigma) colorimetric development. The blot was dried, scanned and slot profiles quantified using Scion Image. Statistical testing (student’s t-test) was performed in Origin 8.0. The entire experiment was repeated twice.

Gu L, & Robinson R.A. (2016). High-throughput Endogenous Measurement of S-Nitrosylation in Alzheimer’s Disease using Oxidized Cysteine-selective cPILOT. The Analyst, 141(12), 3904-3915.

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Profiling Mycobacterium tuberculosis Virulence Factors

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H37Rv lysates and culture filtrate protein (CFP) were prepared as previously described Correa et al. (2014) (link). Bacterial cultures (200 mL) were grown in Sauton media to late log phase and then pelleted by centrifugation at 2,000 × g for 20 min. Supernatant was transferred to a fresh tube, centrifuged again, and then filtered through a 0.2 μm filter unit. Supernatant was concentrated by protein acetone precipitation. Bacterial pellet was used for H37Rv lysates by sonication. Samples were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% powdered milk in PBS and probed with the anti-MtLAP or anti-Zmp1 polyclonal serum produced in mice, followed by incubation with the anti-mouse IgG-alkaline phosphatase secondary antibody (Sigma–Aldrich). Antigen-antibody complex was detected via BCIP/NBT liquid substrate (Sigma–Aldrich).

Correa A.F., Bastos I.M., Neves D., Kipnis A., Junqueira-Kipnis A.P, & de Santana J.M. (2017). The Activity of a Hexameric M17 Metallo-Aminopeptidase Is Associated With Survival of Mycobacterium tuberculosis. Frontiers in Microbiology, 8, 504.

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