Bradford assay method

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The Bradford assay method is a colorimetric technique used to measure the concentration of protein in a solution. It involves the binding of the dye Coomassie Brilliant Blue G-250 to proteins, resulting in a color change that can be measured spectrophotometrically. The Bradford assay provides a simple, rapid, and sensitive way to quantify protein levels in various samples.

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Image lab 5, by Bio-Rad (3 mentions) Chemidoc touch imaging system, by Bio-Rad (3 mentions) Hek293, by American Type Culture Collection (3 mentions) Iscove s modified dulbecco medium, by Thermo Fisher Scientific (2 mentions) Rnase a, by Merck Group (1 mentions) Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions) Chelex resin, by Merck Group (1 mentions) Image lab software, by Bio-Rad (1 mentions) Ecl plus, by GE Healthcare (1 mentions) Citrullinated histone h3 cith3, by Abcam (1 mentions) Nf κb, by Santa Cruz Biotechnology (1 mentions) Stain free technology, by Bio-Rad (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Hrp conjugated igg secondary antibody, by Santa Cruz Biotechnology (1 mentions) Gradient gel, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Ab13847, by Abcam (1 mentions) Ab32503, by Abcam (1 mentions)

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Bradford assay (3878 citations) Bradford method (802 citations) Bradford protein assay method (14 citations) Bradford-based method protein assay (3 citations) Bradford method assay kit (2 citations)

16 protocols using bradford assay method

Protein Extraction and Western Blot Analysis

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Cells were extracted with 1% NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.5 10 mM EDTA, 10 mM NaF, 10 mM Na₄P₂O₇, 0.4 mM Na₃VO₄) with freshly added protease inhibitors (10 μg/ml leupeptin, 4 μg/ml pepstatin and 0.1 Unit/ml aprotinin). Lysates were centrifuged at 13 000 × g for 10 minutes at 4 °C and the supernatants were collected and assayed for protein concentration with the Bradford assay method (Bio-Rad).
Proteins were separated by SDS-PAGE under reducing conditions. Following SDS-PAGE, proteins were transferred to nitrocellulose, reacted with specific antibodies and then detected with peroxidase-conjugate secondary antibodies and chemioluminescent ECL reagent. Digital images were taken with the Bio-Rad ChemiDoc Touch Imaging System and quantified using Bio-Rad Image Lab 5.2.1.

Zonca S., Pinton G., Wang Z., Soluri M.F., Tavian D., Griffin M., Sblattero D, & Moro L. (2017). Tissue transglutaminase (TG2) enables survival of human malignant pleural mesothelioma cells in hypoxia. Cell Death & Disease, 8(2), e2592-.

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Preparation of Nuclear Extracts from RAW 264.7 Cells

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Nuclear extracts from cultured RAW 264.7 cells were prepared using a nuclear protein kit (Millipore, Billerica, MA). The protocol provided by the manufacturer was followed. Protein concentration was measured by the Bradford assay method (BioRad, Hercules, CA). Nuclear extracts were stored in liquid nitrogen till using.

Wang X., Bu H.F., Liu S.X., De Plaen I.G, & Tan X.D. (2015). Molecular mechanisms underlying the regulation of the MFG-E8 gene promoter activity in physiological and inflammatory conditions. Journal of cellular biochemistry, 116(9), 1867-1879.

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Immunoprecipitation and Western Blot Analysis

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HEK293T cells were transfected with indicated plasmids for 30 h and homogenized in ice-cold lysis buffer (50 mmol/L Tris-HCl [pH 7.4], 1% Triton X-100, 150 mmol/L NaCl, 1 mmol/L EDTA [pH 7.4], 0.1% sodium dodecyl sulfate [SDS] and protease inhibitor cocktail). The supernatants were collected via centrifugation and treated with or without RNase A (Sigma-Aldrich), then incubated with mouse anti-HA or Flag antibodies and Protein A/G Magnetic Beads (MCE) according to the manufacturer's instructions and analyzed via WB.
For WB, cells were harvested and homogenized in ice-cold lysis buffer at 4 °C for 30 min. The supernatants were collected via centrifugation at 12,000×g at 4 °C for 30 min, and determined protein concentrations using the Bradford assay method (Bio-Rad). Samples were boiled with SDS-PAGE loading buffer at 100 °C for 10 min and resolved via 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to nitrocellulose membranes. The membrane was blocked with 5% milk in PBS with 0.1% Tween 20 (PBST) before being incubated with primary antibodies and HRP-conjugated goat anti-mouse or -rabbit IgG secondary antibodies (Thermo Fisher Scientific). The results were detected on a Fujifilm LAS-4000 imaging system.

Zhang Q., Ye H., Liu C., Zhou H., He M., Liang X., Zhou Y., Wang K., Qin Y., Li Z, & Chen M. (2023). PABP-driven secondary condensed phase within RSV inclusion bodies activates viral mRNAs for ribosomal recruitment. Virologica Sinica, 39(2), 235-250.

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Protein Analysis Using NP-40 Lysis

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For protein analysis, cells were extracted with 1% NP-40 lysis buffer (50 mM Tris-HCl pH 8.5 containing 1% NP-40, 150 mM NaCl, 10 mM EDTA, 10 mM NaF, 10 mM Na₄P₂O₇, and 0.4 mM Na₃VO₄) with freshly added protease inhibitors (10 μg mL⁻¹ leupeptin, 4 μg mL⁻¹ pepstatin, and 0.1 U mL⁻¹ aprotinin). Lysates were centrifuged at 13,000× g for 10 min at 4 °C and the supernatants were collected and assayed for protein concentration with the Bradford assay method (Bio-Rad). Proteins were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing conditions. Following SDS-PAGE, proteins were transferred to nitrocellulose, reacted with specific antibodies, and then detected with peroxidase-conjugate secondary antibodies and chemioluminescent ECL reagent. Digital images were taken with the Bio-Rad ChemiDocTM Touch Imaging System and quantified using Bio-Rad Image Lab 5.2.1.

Gabano E., Pinton G., Balzano C., Boumya S., Osella D., Moro L, & Ravera M. (2021). Unsymmetric Cisplatin-Based Pt(IV) Conjugates Containing a PARP-1 Inhibitor Pharmacophore Tested on Malignant Pleural Mesothelioma Cell Lines. Molecules, 26(16), 4740.

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Cell Culture Conditions and Protein Analysis

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Cell lines HEK-293 (ATCC CRL 1573), HeLa (ATCC CCL 2) and 3T3 (ATCC CRL 1658), as well as the Phoenix packaging cells (Orbigen, Inc., San Diego, CA, USA), were grown in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific/Gibco, Waltham, MA, USA) supplemented with 10% fetal calf serum, and penicillin/streptomycin. HoxB4 cells (kindly provided by Drs Simona Saccani and Dominic van Essen and described in Stump et al.^{32 (link)}) were grown in Iscove’s Modified Dulbecco Medium (Thermo Fisher Scientific/Gibco) supplemented with 2% fetal calf serum, 1% interleukin 3, 0.5% interleukin 6, 20 ng/ml stem cell factor, 0.3 mg/l peptone, 5 mg/l insulin, nonessential amino acids, β-mercaptoethanol and penicillin/streptomycin. Cells were grown at 37 °C with 5% CO₂. For western blot and coimmunoprecipitation experiments, whole-cell lysates were prepared as previously described.^{31 (link)} Protein concentrations were determined using the Bradford assay method (Bio-Rad, Hercules, CA, USA). Details on the retroviral infection of HoxB4 cells are provided in Supplementary Information.

Thiel V.N., Giaimo B.D., Schwarz P., Soller K., Vas V., Bartkuhn M., Blätte T.J., Döhner K., Bullinger L., Borggrefe T., Geiger H, & Oswald F. (2017). Heterodimerization of AML1/ETO with CBFβ is required for leukemogenesis but not for myeloproliferation. Leukemia, 31(11), 2491-2502.

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Purification of Protein N-Terminal Regions

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For purification of the N-terminal region (N^ter) of proteins (the first 51 amino acid residues), we used the pMal™ Protein Fusion and Purification System (Biolabs). The llmg_0524’ fragment was amplified by PCR and cloned into the pMal-c4x vector and the product established into E. coli strain TG1. A transformant containing the plasmid pMal-0524^Nter was cultured in SOC medium supplemented or not with zinc chloride 0.1 mM. When OD₆₀₀ was close to 0.5 we added IPTG (isopropyl 1-thiol-β-D-galactopyranoside, 0.5 mM). After 4 h of incubation, cells were collected and washed twice in 10 mM Tris–HCl pH 7.5 buffer, pretreated with Chelex resin (Sigma), and stored as a cell pellet at −80 °C. We purified MalE-0524^Nter by affinity chromatography according to manufacter’s procedure (Biolabs) [51 ] with Chelex-treated buffers. The same strategy was used to produce the fusion MalE-0526^Nter. Protein purity was confirmed by SDS-PAGE and proteins were concentrated in spin columns (Centricon, Amicon, cut off: 10 KDa). Protein concentrations were determined with the Bradford assay method (Biorad) with bovine serum albumin as the standard.

Roussel C., Cesselin B., Cachon R, & Gaudu P. (2015). Characterization of two Lactococcus lactis zinc membrane proteins, Llmg_0524 and Llmg_0526, and role of Llmg_0524 in cell wall integrity. BMC Microbiology, 15, 246.

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Quantification of Citrullinated Histone H3

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Cytoplasmic and nuclear extracts were prepared according to standard protocols [36 (link)] and the Bradford assay method (Bio-Rad) was used to determine protein concentration. Then, 20 μg of both cytoplasmic and nuclear extracts were separated by SDS-PAGE. Antibodies against PAD4 (1:1000; Abcam), citrullinated histone H3 (CitH3) (1:1000; Abcam) and nuclear factor (NF)-κB (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were dispensed overnight (4 °C) and peroxidase-conjugated secondary antibody was administered for 1 h (RT). The immunoreaction was visualized using ECL plus (GE Healthcare, Buckinghamshire, UK). Stain-free technology (Bio-Rad) was used as loading protein control and densitometric analysis was performed with Image Lab software (Bio-Rad).

Ruiz-Limon P., Ladehesa-Pineda M.L., Castro-Villegas M.D., Abalos-Aguilera M.D., Lopez-Medina C., Lopez-Pedrera C., Barbarroja N., Espejo-Peralbo D., Gonzalez-Reyes J.A., Villalba J.M., Perez-Sanchez C., Escudero-Contreras A., Collantes-Estevez E., Font-Ugalde P, & Jimenez-Gomez Y. (2020). Enhanced NETosis generation in radiographic axial spondyloarthritis: utility as biomarker for disease activity and anti-TNF-α therapy effectiveness. Journal of Biomedical Science, 27, 54.

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Protein Extraction from Cell Lines

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Cell lines HEK293 (ATCC CRL 1573) and HeLa (ATCC CCL 2) were cultivated in DMEM supplemented with 10% FCS, and Penicillin/Streptomycin. For Western blotting and immunoprecipitation experiments whole-cell lysates were prepared essentially as previously described⁶⁶. Protein concentrations were determined using the Bradford assay method (BioRad).

Philippi A., Heller S., Costa I.G., Senée V., Breunig M., Li Z., Kwon G., Russell R., Illing A., Lin Q., Hohwieler M., Degavre A., Zalloua P., Liebau S., Schuster M., Krumm J., Zhang X., Geusz R., Benthuysen J.R., Wang A., Chiou J., Gaulton K., Neubauer H., Simon E., Klein T., Wagner M., Nair G., Besse C., Dandine-Roulland C., Olaso R., Deleuze J.F., Kuster B., Hebrok M., Seufferlein T., Sander M., Boehm B.O., Oswald F., Nicolino M., Julier C, & Kleger A. (2021). ONECUT1 mutations and variants in diabetes. Nature medicine, 27(11), 1928-1940.

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Extracellular Vesicle Protein Extraction

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EVs were pelleted by centrifugation at 100,000 × g for 1 h at +4 °C, supernatants were discarded and proteins were extracted from the pellet using RIPA buffer. Cell layers were scrapped in RIPA buffer. Both lysates with the Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Illkirch, France) and scraped. Lysates were mechanically resuspended by pipetting and vortexing every 5 min for 30 min. They were then centrifuged at 10,000 × g for 10 min at +4 °C to discard insoluble protein and the amount of soluble protein was quantified by Bradford assay method (Bio-Rad) using bovine serum albumin (Sigma) as a standard.

Brassart B., Da Silva J., Donet M., Seurat E., Hague F., Terryn C., Velard F., Michel J., Ouadid-Ahidouch H., Monboisse J.C., Hinek A., Maquart F.X., Ramont L, & Brassart-Pasco S. (2019). Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA. British Journal of Cancer, 120(4), 453-465.

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Murine T-cell culture and inhibition

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Murine pre-T lymphoma cell line (Beko) and murine hybridoma mature T-cell line (MT) were grown in Iscove's Modified Dulbecco Medium (Gibco) supplemented with 2% FCS, 0.3 mg/l peptone, 5 mg/l insulin, nonessential aminoacids and penicillin/streptomycin. Cells were grown at 37°C with 5% CO₂. pre-T cells were treated with 10 μg/ml DAPT/GSI (Alexis ALX-270–416-M025) or DMSO as control. Mature T-cells were treated with 25 μM TBB (4,5,6,7-tetrabromobenzotriazole, Sigma-Aldrich T0826), 25 μM TBCA [(E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid, Millipore 218710] or DMSO as control. Cell lines HEK293 (ATCC CRL 1573), 293 T and HeLa (ATCC CCL 2) were cultivated in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin.
For western blotting, EMSA and immunoprecipitation experiments whole-cell lysates were prepared as previously described (20 (link)). Protein concentrations were determined using the Bradford assay method (BioRad).

Oswald F., Rodriguez P., Giaimo B.D., Antonello Z.A., Mira L., Mittler G., Thiel V.N., Collins K.J., Tabaja N., Cizelsky W., Rothe M., Kühl S.J., Kühl M., Ferrante F., Hein K., Kovall R.A., Dominguez M, & Borggrefe T. (2016). A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes. Nucleic Acids Research, 44(10), 4703-4720.

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