Bradford assay

Manufactured by Cytoskeleton

Sourced in United States

The Bradford assay is a colorimetric protein quantification method. It is used to determine the concentration of protein in a solution by measuring the absorbance of the sample at a specific wavelength.

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Lab products found in correlation

Quantity one software package, by Bio-Rad (1 mentions) Horseradish peroxidase conjugated goat anti mouse secondary antibody, by Bio-Rad (1 mentions) 0.22 m pore size filter, by Merck Group (1 mentions) N2a cells, by American Type Culture Collection (1 mentions) Horse serum, by Merck Group (1 mentions) Pc12 cells, by American Type Culture Collection (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Dulbecco s modified eagle s medium, by Merck Group (1 mentions) Complete mini protease inhibitor cocktail, by Roche (1 mentions) Nerve growth factor (ngf), by Alomone (1 mentions)

4 protocols using bradford assay

Loa loa Antigen Extraction and Administration

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LsAg was prepared as described previously (33 (link)). In brief, L. sigmodontis adult worms were harvested from infected gerbils' thoracic cavities and mechanically homogenized on ice in endotoxin-free PBS (PAA; Pasching, Austria). The supernatant was collected and protein quantification was done by Bradford assay (Cytoskeleton; Denver, CO., USA). Aliquots of LsAg were stored for later usage at −80°C.
LsAg treatment was performed as previously described (19 (link)). Daily i.p. injections of 2 μg LsAg per mouse for 2 weeks were given to obese mice during weeks 14–16 of HFD. Corresponding control mice received PBS injections. After the final LsAg injection, the GTT and immunological studies were performed.

Surendar J., Frohberger S.J., Karunakaran I., Schmitt V., Stamminger W., Neumann A.L., Wilhelm C., Hoerauf A, & Hübner M.P. (2019). Adiponectin Limits IFN-γ and IL-17 Producing CD4 T Cells in Obesity by Restraining Cell Intrinsic Glycolysis. Frontiers in Immunology, 10, 2555.

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Protein Expression Analysis in Transfected Cells

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2.5 × 10⁵ cells were plated on 6 well plates and transfected as indicated above. Cells were harvested at ~60 % confluency at 24 h post-transfection by scraping into lysis buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4, 10 mM EDTA, 0.6 % NP-40, and the complete mini protease inhibitor cocktail (Roche)). Nuclei were removed by pelleting at 20,000 g for 5 min. Protein concentrations were determined by Bradford assay (Cytoskeleton, USA). Fifteen micrograms of protein was resolved on an SDS-polyacrylamide gel and electrotransferred to a polyvinylidene diflu- oride (PVDF) membrane. Antibodies used in this study were anti-hemagglutinin (anti-HA; clone 16B12; Covance), and a goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Blots were developed with the SuperSignal substrate (Thermo Scientific) and quantified using the Quantity One software package (Bio-Rad).

Dieudonné F.X., O’Connor P.B., Gubler-Jaquier P., Yasrebi H., Conne B., Nikolaev S., Antonarakis S., Baranov P.V, & Curran J. (2015). The effect of heterogeneous Transcription Start Sites (TSS) on the translatome: implications for the mammalian cellular phenotype. BMC Genomics, 16, 986.

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Extraction of Parasitic Antigens

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Adult worms, L3, L4 and L5 were thawed and concentrated by centrifuging at 1500× rpm for 5 min using a bench-top centrifuge (Human Biochemica und Diagnostica GmbH, Wiesbaden, Germany). Parasites were then mechanically minced on ice in cold sterile endotoxin-free PBS (Sigma-Aldrich). Insoluble material was removed by centrifugation at 2000× rpm for 10 min at 4 °C. The resulting soluble parasite antigen extract was then filtered through a 0.22-µm pore size filter (Merck Millipore) and protein concentration was determined by a Bradford assay (Cytoskeleton, Denver, USA) according to manufacturer’s description. Aliquots were frozen at − 80 °C until required.

Chunda V.C., Ritter M., Bate A., Gandjui N.V., Esum M.E., Fombad F.F., Njouendou A.J., Ndongmo P.W., Taylor M.J., Hoerauf A., Layland L.E., Turner J.D, & Wanji S. (2020). Comparison of immune responses to Loa loa stage-specific antigen extracts in Loa loa-exposed BALB/c mice upon clearance of infection. Parasites & Vectors, 13, 51.

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Cell Culture and Transfection Protocols

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HEK293T (ATTC, CRL-11268) and N2a cells (ATCC, CCL-131) were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% foetal calf serum (FCS) (Brunschwig), 1% penicillin/streptomycin (Gibco), in a humidified atmosphere containing 5% CO₂. PC12 cells (ATCC, CRL-1721) were grown on collagen coated plates in RPMI-1640 medium supplemented with 10% horse serum (Sigma), 5% FCS and 1% penicillin/streptomycin. For differentiation, neuronal growth factor (NGF, Alomone labs) was added at 50 ng/mL under the serum conditions indicated in the text. Neurite outgrowth was visible three days later. N2a cells were transfected using Lipofectamine 2000 (Life Technologies). Transfections were performed in normal growth medium when the cells were no more than 50% confluent. Four hours later the medium was replaced with fresh growth medium except in those experiments in which the serum concentration was altered. Cytoplasmic extracts were prepared by solubilizing the monolayer in 150 mM NaCl, 50 mM Tris–HCl pH 7.4, 10 mM EDTA, 0.6% NP-40, and the complete mini protease inhibitor cocktail (Roche). Nuclei were removed by pelleting at 20,000 g for 5 min. Protein concentrations were determined by Bradford assay (Cytoskeleton, USA). Transfections were generally carried out at least in duplicate and repeated at least twice.

Legrand N., Araud T., Conne B., Kuijpers O., Jaquier-Gubler P, & Curran J. (2014). An AUG Codon Conserved for Protein Function Rather than Translational Initiation: The Story of the Protein sElk1. PLoS ONE, 9(7), e102890.

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