Bradford protein assay dye reagent

Manufactured by Bio-Rad

Sourced in United States

The Bradford protein assay dye reagent is a laboratory product used to quantify the total protein concentration in a sample. It is a colorimetric assay that measures the binding of the dye Coomassie Brilliant Blue G-250 to proteins, resulting in a color change that can be measured spectrophotometrically.

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Bradford assay (3878 citations) Bio-Rad protein assay (2654 citations) Bradford protein assay (1622 citations) Bradford reagent (1210 citations) Protein assay reagent (895 citations) Protein Assay Dye Reagent (425 citations) Protein assay dye (194 citations) Bradford protein assay reagent (136 citations) Bradford assay reagent (100 citations) Dye reagent (53 citations)

18 protocols using bradford protein assay dye reagent

Agarose Microbead-Based Enzyme Assay

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Agarose microbeads (50–150 μm
diameter) were purchased from Agarose Bead Technologies (Madrid, Spain).
Polyethyleneimine (PEI) solution in H₂O (M_w ∼ 60 000, 50 wt %), polyallylamine (PAH)
solution in H₂O (M_w ∼
65 000, 10 wt %), polydiallyldimethylammonium chloride (PDADMAC)
solution in H₂O (M_w < 100 000,
35 wt %), pyridoxal 5′-phosphate hydrate (PLP, 98%), rhodamine
B isothiocyanate mixed isomers (RhB), acetone, 2-phenylethylamine
(PEA, 98%), iminodiacetic acid (IDA), albumin bovine serum standard
(BSA), and other reagents and solvents of analytical grade were purchased
from Sigma-Aldrich (St. Louis, IL). Nicotinamide adenine dinucleotide-reduced
sodium salt (NADH) and flavin mononucleotide sodium salt (FMN) were
purchased from GERBU Biotechnik GmbH (Heidelberg, Germany). Flavin
Adenine dinucleotide disodium salt hydrate (FAD, 94%) was purchased
from Cymit Quimica S.L. (Barcelona, Spain). Riboflavin (Rf, 98%) was
purchased from Acros Organics B.V.B.A. (Fair Lawn, New Jersey, United
States). Isopropyl-β-d-thiogalactopiranoside (IPTG,
100%) was purchased from Fisher Bioreagents. The Bradford protein
assay dye reagent was purchased from BIORAD (Biorad. Hercules, CA).
Clear bottom black and white microplates (96-well) were purchased
from Avantor (2021 VWR International, LLC). μ-Slides 8 well
glass bottom was purchased from Ibidi (Planegg, Germany).

Diamanti E., Santiago-Arcos J., Grajales-Hernández D., Czarnievicz N., Comino N., Llarena I., Di Silvio D., Cortajarena A.L, & López-Gallego F. (2021). Intraparticle Kinetics Unveil Crowding and Enzyme Distribution Effects on the Performance of Cofactor-Dependent Heterogeneous Biocatalysts. ACS Catalysis, 11(24), 15051-15067.

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Expression and Purification of TS Enzyme

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The pure (E,E)‐FPP standard was purchased from Isoprenoids (Tampa, Florida, USA) and used for all TS assays reported. Gene sequences were optimized for codon usage in E. coli and purchased from GeneArt (ThermoFisher Scientific, Paisley, UK). DNA‐modifying enzymes were purchased from Life Technologies (Paisley, UK). Bradford protein assay dye reagent was obtained from Bio‐Rad (Hertfordshire, UK). All other chemicals were purchased from Sigma‐Aldrich except where indicated otherwise.

Styles M.Q., Nesbitt E.A., Marr S., Hutchby M, & Leak D.J. (2017). Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes. The Febs Journal, 284(11), 1700-1711.

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Protein Quantification using Bradford Assay

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BSA (M_w 66 kg/mol), HPβCD (average M_w ~ 1540 g/mol), sodium phosphate, and Coomassie brilliant blue R were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bradford protein assay dye reagent and acrylamide were acquired from Bio-Rad Laboratories (Hercules, CA, USA). Glacial acetic acid and ortho-phosphoric acid were obtained from Merck KGaA (Darmstadt, Germany). PageRuler™ prestained protein ladder (10 to 180 kDa) was purchased from Thermo Scientific (Waltham, MA, USA). Methanol was obtained from Anaqua Global International (Cleveland, OH, USA). Ultrapure water used was purified by Barnstead NANOpure Diamond™ water system with a 0.2 µm filter (APS Water Services, Van Nuys, CA, USA). All solvents and reagents were of analytical grade or better unless otherwise specified.

Lo J.C., Pan H.W, & Lam J.K. (2021). Inhalable Protein Powder Prepared by Spray-Freeze-Drying Using Hydroxypropyl-β-Cyclodextrin as Excipient. Pharmaceutics, 13(5), 615.

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Western Blot Analysis of Skeletal Muscle

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For Western blotting, skeletal muscles were frozen in liquid nitrogen and ground with a mortar and pestle. Ground tissues were then lysed in RIPA buffer containing protease and phosphatase inhibitors as previously described [35 (link)]. Lysates were then cleared by centrifugation at 14,000RPM for 5 min at 4 °C. Protein concentration was determined by using Bradford protein assay dye reagent (Bio-Rad, Mississauga, ON, Canada). Equal amounts of protein were run on polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (PVDF). Membranes were probed with primary antibodies for 1 h at room temperature, or overnight at 4 °C followed by secondary HRP-conjugated antibodies for 1 h at room temperature. Antibody detection was performed by chemiluminescence and X-ray film exposure.

Pryce B.R., Al-Zahrani K.N., Dufresne S., Belkina N., Labrèche C., Patino-Lopez G., Frenette J., Shaw S, & Sabourin L.A. (2017). Deletion of the Ste20-like kinase SLK in skeletal muscle results in a progressive myopathy and muscle weakness. Skeletal Muscle, 7, 3.

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Western Blot Analysis of PLCγ and cGAS

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Adherent cells were washed with ice-cold PBS and lysed and scrapped in CelLytic^™ M buffer (Sigma) with 1X Halt^™ protease and phosphatase cocktail inhibitors (Thermo Fisher) before being centrifuged for 15 minutes at 20,000 x g at 4°C. Proteins were harvested from supernatant and quantified by using Bradford protein assay dye reagent (Bio-Rad). 25 μg of protein from whole cell extract were loaded in 4–10% acrylamide gels and transferred on nitrocellulose membranes. Membranes were stained overnight in 5% BSA-containing PBS Tween 0.1% with following antibodies: mouse anti-PLCγ (1:2000; Millipore #05–163) and rabbit anti-cGAS (mouse; 1:1000; Cell signaling #31659). Proteins were revealed with fluorescent secondary anti-rabbit (1:10000; LI-COR #926–68073) or anti-mouse antibodies (1:10000; LI-COR #926–32212) using the LI-COR fluorescent scanner.

Jacoberger-Foissac C., Cousineau I., Bareche Y., Allard D., Chrobak P., Allard B., Pommey S., Messaoudi N., McNicoll Y., Soucy G., Koseoglu S., Masia R., Lake A.C., Seo H., Eeles C.B., Rohatgi N., Robson S.C., Turcotte S., Haibe-Kains B, & Stagg J. (2023). CD73 inhibits cGAS-STING and cooperates with CD39 to promote pancreatic cancer. Cancer immunology research, 11(1), 56-71.

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Decylubiquinol Synthesis and Quantification

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P. falciparum 3D7 strain were obtained from the Liverpool School of Tropical Medicine. Protease cocktail inhibitor was obtained from Roche. Bradford protein assay dye reagent was obtained from Bio-Rad. All other reagents were obtained from Sigma-Aldrich. Decylubiquinol was produced as per Fisher et al. (2009 (link)). In brief, 25 mg of decylubiquinone were dissolved in 400 μL of nitrogen-saturated hexane. An equal volume of aqueous 1 M sodium dithionite was added, and the mixture vortexed until colorless. The organic phase containing the decylubiquinol was collected, the solvent was evaporated under N₂ and the decylubiquinol finally dissolved in 100 μL of 96% ethanol (acidified with 10 mM HCl). Concentrations of decylubiquinol was determined spectrophotometrically on a Cary 300 Bio UV/visible spectrophotometer (Varian, UK) from absolute spectra, using ε_288−−320 = 8.1 mM⁻¹.cm⁻¹. Decylubiquinol was stored at −80°C and used within 2 weeks.

McPhillie M.J., Zhou Y., Hickman M.R., Gordon J.A., Weber C.R., Li Q., Lee P.J., Amporndanai K., Johnson R.M., Darby H., Woods S., Li Z.H., Priestley R.S., Ristroph K.D., Biering S.B., El Bissati K., Hwang S., Hakim F.E., Dovgin S.M., Lykins J.D., Roberts L., Hargrave K., Cong H., Sinai A.P., Muench S.P., Dubey J.P., Prud'homme R.K., Lorenzi H.A., Biagini G.A., Moreno S.N., Roberts C.W., Antonyuk S.V., Fishwick C.W, & McLeod R. (2020). Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites. Frontiers in Cellular and Infection Microbiology, 10, 203.

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Protein Analysis of RAW 264.7 Cells and Murine Colon

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RAW 264.7 cells were cultured as described above, and the cell lysate was prepared using an ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail and phenylmethane sulfonyl fluoride (Sigma-Aldrich). Mice colon samples were homogenized using the Bio-Masher II (Optima, Tokyo, Japan). The protein concentration was measured using the Bradford Protein Assay Dye Reagent (Bio-Rad, Hercules, CA, USA), and the total cell lysate, nuclear and cytosolic lysates were used for Western blot analysis as previously described [24 (link)]. The antibodies used were: β-actin, Lamin B, cyclooxygenase-2 (Cox2), heme oxygenase-1 (HO-1) (Santa Cruz Biotechnology, Dallas, TX, USA); nuclear factor-erythroid factor 2-related factor 2 (Nrf2) (Abcam, Cambridge, MA, USA); phospho-nuclear factor-κB (p-NF-κB), NF-κB, p-IκBα, TNF-α, inducible nitric oxide synthase (iNOS) (Cell Signaling Technology, Danvers, MA, USA). Anti-rabbit and anti-mouse secondary antibodies were obtained from Santa Cruz (USA). Proteins were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and detected using the LAS 4000 with Multi Gauge 3.1 software (Fujifilm, Tokyo, Japan). The protein band was measured using ImageJ software (NIH, Bethesda, MD, USA).

Shin J.M., Son Y.J., Ha I.J., Erdenebileg S., Jung D.S., Song D.G., Kim Y.S., Kim S.M, & Nho C.W. (2022). Artemisia argyi extract alleviates inflammation in a DSS-induced colitis mouse model and enhances immunomodulatory effects in lymphoid tissues. BMC Complementary Medicine and Therapies, 22, 64.

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Pulse-chase analysis of protein degradation in cardiomyocytes

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Protein degradation protocol was adapted from ref. ^{38 (link)} for use with HPG. Twenty-four hours after treatment with PE/Colchicine, NRVMs were methionine depleted and labeled with HPG for 4 h. After labeling, the NRVMs were washed in serum-free culture media containing excess methionine (3 mM, Sigma-Aldrich) three times at 30-min intervals. Cells were collected in 1X RIPA lysis buffer (Cayman Chemical) + 1X Protease/Phosphatase Inhibitor Cocktail (Cell Signaling) at 0, 1, 2, and 3 days post-labeling. Colchicine or PE treatment was maintained throughout the pulse/chase steps. Protein concentration was determined by Bradford protein assay dye reagent (Bio-Rad) and 25 µg of total lysate per sample were labeled with 4 nmol of IRDye 800CW Azide Infrared dye (LI-COR Biosciences) using the ClickiT Protein Reaction Buffer kit (Thermo Fisher Scientific).

Scarborough E.A., Uchida K., Vogel M., Erlitzki N., Iyer M., Phyo S.A., Bogush A., Kehat I, & Prosser B.L. (2021). Microtubules orchestrate local translation to enable cardiac growth. Nature Communications, 12, 1547.

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Bradford Assay for Protein Quantification

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To determine the protein concentration in EPS fractions, we used the Bradford protein assay in microtiter 96-well plates. Aliquots of 20 μL EPS were poured into the wells and mixed with 180 μL Bradford Protein Assay Dye Reagent (Bio-Rad). The mixture was incubated for 5 min, and the Abs (595 nm) was measured by a Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA).

Minich A., Levarski Z., Mikulášová M., Straka M., Liptáková A, & Stuchlík S. (2022). Complex Analysis of Vanillin and Syringic Acid as Natural Antimicrobial Agents against Staphylococcus epidermidis Biofilms. International Journal of Molecular Sciences, 23(3), 1816.

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Tumor Protein Extraction and Analysis

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Snap-frozen tumors were subjected to cryogenic grinding using a CyroMill (Retsch). Resulting tumor lysate or whole cell pellets were lysed on ice in cold NETN buffer (0.5% NP-40, 1 mM EDTA, 150 mM NaCl and 20 mM Tris Buffer) supplemented with cOmplete, Mini EDTA-free protease inhibitor cocktail (Millipore Sigma; Catalog No. 11836170001) and phosphatase inhibitor cocktail 2 and 3 (Sigma Aldrich; Catalog No. P5726 and Catalog No. P0044), then sonicated (Branson 450 Sonifier). Protein was quantified using Bradford Protein Assay Dye Reagent (Bio-Rad; Catalog No. 5000006). Western blots were run on 15 – 20 μg protein per lane, and then imaged and quantified as we previously described (5 (link),33 (link)). Antibodies used were anti-GAPDH (Millipore #MAB374), anti-Vinculin (Santa Cruz SC-73614), anti-DNA-PKcs (Santa Cruz SC-5282), anti-P-S2056-DNA-PKcs (Abcam ab18192), anti-AKT1 (Cell Signaling Technology (CST) #2938), anti-P-S473-AKT (CST #4060), anti-P-Th308-AKT (CST #4056), anti-P-S139-H2AX (AbCam #26350), anti-PARP1 (CST #9542), anti-P-S235/236-S6 (CST #4858) and anti-S6 (CST #2217).

Castellano G.M., Zeeshan S., Garbuzenko O.B., Sabaawy H.E., Malhotra J., Minko T, & Pine S.R. (2022). Inhibition of mTORC1/2 and DNA-PK via CC-115 Synergizes with Carboplatin and Paclitaxel in Lung Squamous Cell Carcinoma. Molecular cancer therapeutics, 21(9), 1381-1392.

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