Bradford assay

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The Bradford assay is a colorimetric protein assay used to measure the concentration of protein in a solution. It is based on the color change of the Coomassie Brilliant Blue G-250 dye in response to various concentrations of protein.

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Lab products found in correlation

Pvdf membrane, by Merck Group (133 mentions) Protease inhibitor cocktail, by Merck Group (106 mentions) Protease inhibitor cocktail, by Roche (88 mentions) Nitrocellulose membrane, by Bio-Rad (73 mentions) Protease inhibitor, by Roche (67 mentions) Pvdf membrane, by Bio-Rad (66 mentions) β actin, by Merck Group (60 mentions) Ripa buffer, by Thermo Fisher Scientific (54 mentions) Complete protease inhibitor cocktail, by Roche (49 mentions) Ripa buffer, by Merck Group (48 mentions) Polyvinylidene difluoride membrane, by Merck Group (47 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (44 mentions) β actin, by Cell Signaling Technology (42 mentions) Bovine serum albumin (bsa), by Merck Group (41 mentions) Protease inhibitor, by Merck Group (37 mentions) Image lab software, by Bio-Rad (33 mentions) β actin, by Santa Cruz Biotechnology (33 mentions) Nitrocellulose membrane, by GE Healthcare (32 mentions) Gapdh, by Cell Signaling Technology (31 mentions) Anti β actin, by Merck Group (30 mentions)

Close spelling variants of this product

Bradford assay protocol Quick Star Bradford protein assay Bradford quantification assay Bradford Biorad Assay Bradford assay (Protein Assay Dye Reagent Bradford DC assay Bradford-based assay DC Bradford assay kit Bradford's assay kit Bradford protein assay solution

3 878 protocols using bradford assay

Quantifying Histone H3K27 Methylation

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Total levels of trimethyl Lys27 on histone H3 was quantified using a H3K27me3 ELISA (Active Motif). Briefly, acid extracted histones from WT and KI mouse Schwann cells were prepared using manufacturer's directions and protein concentrations were calculated using a Bradford Assay (Biorad). Equal amounts of histone extracts were added to the plate and the sandwich ELISA assay was conducted following the manufacturer's protocol.
Histone methyltransferase activity was quantified in WT and KI nuclear extracts from mouse Schwann cells. Nuclear extracts were isolated using Abcam's Nuclear Extraction Kit and protein concentrations were calculated using a Bradford Assay (Biorad). Equal amounts of nuclear extract protein were analyzed using Histone Methyltransferase H3K27 Activity Quantification Assay Kit (Abcam) and following the manufacturer's protocol. Results were normalized to WT activity levels and HMT activity is represented as a percentage of WT activity.

Ness J.K., Skiles A.A., Yap E., Fajardo E.J., Fiser A, & Tapinos N. (2016). Nuc‐ErbB3 regulates H3K27me3 levels and HMT activity to establish epigenetic repression during peripheral myelination. Glia, 64(6), 977-992.

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Mitochondrial Isolation from Fly and Cell Lines

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~200 Flies were homogenized and mitochondria were isolated [26] (link). The final mitochondrial pellet was resuspended in KHE (120 mM KCl, 3 mM HEPES, 5 mM KH₂PO₄, pH 7.2) and kept on ice. Protein was determined by Bradford assay (Biorad).
ZR75-30 cells were harvested from 2 to 4 500 cm² dishes at ~90% confluence, washed three times in ice-cold STE (250 mM sucrose, 5 mM Tris–HCl, 2 mM EGTA, pH 7.4), scraped off, suspended in 50 ml STE and centrifuged for 10 min at 500g. The pellet was resuspended in STE supplemented with 1% (w/v) fatty acid-free bovine serum albumin and homogenized (up to 20 strokes). The suspension was brought to 50 ml and centrifuged for 5 min at 1000g. The supernatant was centrifuged for 10 min at 11,000g. The pellet was resuspended in STE and centrifuged for 10 min at 10,000g. The mitochondrial pellet was resuspended in STE and kept on ice. Protein was determined by Bradford assay (Biorad).

Goncalves R.L., Rothschild D.E., Quinlan C.L., Scott G.K., Benz C.C, & Brand M.D. (2014). Sources of superoxide/H2O2 during mitochondrial proline oxidation. Redox Biology, 2, 901-909.

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Quantifying Inflammatory Factors in Tumor Tissues

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Snap frozen primary tumor and LN samples were mechanically disrupted and treated by lysis solution (Bio-Rad). Protein concentration in lysates was determined using Bradford assay (Bio-Rad). After sonication, samples were centrifuged at 4,500 g for 6 min at 4 °C. Protein concentration in the supernatant was determined using Bradford assay and adjusted to 1000 μg/ml using serum diluent (both Bio-Rad). Amounts of inflammatory factors in tissue lysates (obtained from tissues from mice treated for 5 days and sacrificed 5 days later) were measured by multiplex technology (Bio-Rad).Transforming growth factor (TGF)-β1 was measured using a single plex kit (Millipore) according to manufacturer's protocol.

Gatti L., Sevko A., Cesare M.D., Arrighetti N., Manenti G., Ciusani E., Verderio P., Ciniselli C.M., Cominetti D., Carenini N., Corna E., Zaffaroni N., Rodolfo M., Rivoltini L., Umansky V, & Perego P. (2014). Histone deacetylase inhibitor-temozolomide co-treatment inhibits melanoma growth through suppression of Chemokine (C-C motif) ligand 2-driven signals. Oncotarget, 5(12), 4516-4528.

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Brain Tissue Fractionation for Immunoprecipitation

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For immunoprecipitation, brain tissues (10% w/v) were homogenized in tris lysis buffer [50 mM Tris–Cl (pH 8.0), 0.5% CHAPS, 1% Triton X100, 1 mM DTT, protease, and phosphatase inhibitors], followed by overnight incubation at 4 °C. The samples were centrifuged at 14,000 rpm for 45 min and the resultant supernatant was saved as tris-soluble fraction. The pellet was resuspended in 70% formic acid via sonication and the resultant slurry was incubated at room temperature for 20 min. The supernatant from the subsequent centrifugation was saved as FA-soluble fraction. Proteins from Tris-soluble fraction were quantified by Bradford assay (Bio-Rad, USA) while those from FA-soluble fraction were quantified by measuring the absorbance at 280 nm using Nanodrop Spectrophotometer due to low quantity and incompatibility with the assay reagents (Thermo Fisher Scientific, USA).
For 1D PAGE, brain tissues (10% w/v) were homogenized in urea-thiourea lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, protease, and phosphatase inhibitors), followed by overnight incubation at 4 °C. The samples were centrifuged, and the supernatant was utilized for analysis after quantification using Bradford assay (Bio-Rad, USA).

Noor A., Zafar S., Shafiq M., Younas N., Siegert A., Mann F.A., Kruss S., Schmitz M., Dihazi H., Ferrer I, & Zerr I. (2021). Molecular Profiles of Amyloid-β Proteoforms in Typical and Rapidly Progressive Alzheimer’s Disease. Molecular Neurobiology, 59(1), 17-34.

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Vitamin D Binding Protein Preparation

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The vitamin D binding protein complex (VDBP-Complex) was provided by Chungbuk National University (Korea). Its concentration was measured by Bradford assay (Bio-Rad, USA). It should be stored at 4 °C in the refrigerator.
The vitamin D binding protein (VDBP) was provided by MyBioSource (Cat#MBS568853, USA). It was reconstituted with 250 µL of filtered sterile third distilled water as described in the manual. It should be stored at − 20 °C or below. Its concentration was measured by Bradford assay (Bio-Rad, USA).
VDBP-Complex and VDBP were diluted using NaHCO₃ buffer to proceed with the coating step. Each target was diluted with 0.1 M NaHCO3 (pH 8.6) to a concentration suitable for coating of 20 µg/mL.
Vitamin D (VD) was provided by Merck (Cat#63283-36-3, Germany). It was reconstituted with 238.86 µL 100% EtOH to 10 mM, as described in the manual. It should be stored at − 20 °C.

Kim T., Lee J., Lee J.P., Kim B.N., Kim Y.H., Lee Y.S, & Min J. (2023). Screening of novel peptides that specifically interact with vitamin D bound biocomplex proteins. Scientific Reports, 13, 2116.

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Epigenetic Modulation of Cancer Cells

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Cancer cells were treated with 6a, 6d, 6e, 6h, and 6l at 5 μM for 30 h. SAHA was used as a positive control at the same time and concentration. For protein extraction lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% NP40, 10 mmol/L NaF, 1 mmol/L PMSF, and protease inhibitor cocktail) was used. Samples were then centrifuged at 13,000 rpm for 30 min at 4 °C and protein concentration quantified by Bradford assay (Bio-Rad). For histone extraction, cells were collected and resuspended in triton extraction buffer [TEB; PBS containing 0.5% Triton × 100 (v/v), 2 mmol/L PMSF, 0.02% (w/v) NaN₃], for 10 minutes at 4 °C. Samples were centrifuged at 2,000 rpm for 10 minutes at 4 °C and pellets washed in TEB (half volume). Samples were then resuspended in 0.2 N HCl, and acid histone extraction was carried out overnight at 4 °C. Protein concentration was determined by Bradford assay (Bio-Rad). 35 μg for each protein extract were loaded on 10% polyacrylamide gels and 2 μg of histone extract were instead used on 15% polyacrylamide gel. Samples were then transferred on nitrocellulose membrane (Trans-blot turbo, Biorad catalog: 1704150) and revealed with Anti-Acetylated Tubulin (clone 6–11B-1, Sigma) and Anti-acetyl-histone H3 Antibody (cod: 06599, Millipore). GAPDH (cod: 14C10, Cell Signaling) and H4 (ab31830, Abcam) antibodies were used as loading controls.

Campiani G., Cavella C., Osko J.D., Brindisi M., Relitti N., Brogi S., Saraswati A.P., Federico S., Chemi G., Maramai S., Carullo G., Jaeger B., Carleo A., Benedetti R., Sarno F., Lamponi S., Rottoli P., Bargagli E., Bertucci C., Tedesco D., Herp D., Senger J., Ruberti G., Saccoccia F., Saponara S., Gorelli B., Valoti M., Kennedy B., Sundaramurthi H., Butini S., Jung M., Roach K.M., Altucci L., Bradding P., Christianson D.W., Gemma S, & Prasse A. (2021). Harnessing the role of HDAC6 in Idiopathic Pulmonary Fibrosis: Design, Synthesis, Structural Analysis, and Biological Evaluation of Potent Inhibitors. Journal of medicinal chemistry, 64(14), 9960-9988.

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Protein Extraction from Microbial Cells

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50 OD₆₀₀ equivalents of cells were grown to mid-log phase and then collected and washed at 4°C. Cells were disrupted by glass bead lysis in 500μl of lysis buffer (50mM sodium phosphate buffer pH 7.5, 100 mM NaCl, 1mM DTT, 2mM PMSF, and 5 μg/ml Pepstatin) using a FastPrep-24 (MP Bio). Lysates were centrifuged at 500×g for 5 min at 4°C to remove cell debris. The protein concentration of the supernatant was determined by Bradford assay (Bio-Rad), and normalized lysates were centrifuged at 15000×g for 15 min at 4°C. The supernatant was removed, and pelleted proteins were washed in buffer (50mM sodium phosphate buffer pH 7.5, 25mM NaCl) and collect by a second centrifugation at 15000×g at 4°C for 15 min. The supernatant was removed, and the protein pellet was resuspended in 50μl of wash buffer, and the protein concentration was determined by Bradford assay (Bio-rad). Samples were analyzed in duplicate, and experiments were repeated at least three times.

Holmes W.M., Mannakee B.K., Gutenkunst R.N, & Serio T.R. (2014). Loss of N-terminal Acetylation Suppresses A Prion Phenotype By Modulating Global Protein Folding. Nature communications, 5, 4383.

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Protein Extraction from Microbial Cells

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Holmes W.M., Mannakee B.K., Gutenkunst R.N, & Serio T.R. (2014). Loss of N-terminal Acetylation Suppresses A Prion Phenotype By Modulating Global Protein Folding. Nature communications, 5, 4383.

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Protein Extraction and Quantification from Cells and Adipose Tissue

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Cells were rinsed twice with ice-cold PBS and then lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 1% Triton X-100; 0.5% deoxycholate; 0.1% SDS; 200 μM PMSF; 1 mM DTT, 1 mM Na3VO4; 10 mM NaF), supplemented with mini protease inhibitor cocktail tablet (Roche, Germany). Lysates were centrifuged at 12000 × g, for 15 min at 4 °C. Protein concentration was determined by the Bradford assay (Biorad) and denatured with SDS buffer (0.5 M Tris, 30% glycerol, 10% SDS, 0.6 M DTT, 0.012% bromophenol blue). After heating for 5 min at 95 °C, the samples were frozen at -20 °C until use.
Adipose tissue was sonicated at 4 °C in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 1% Triton X-100; 0.5% deoxycholate; 0.1% SDS; 200 μM PMSF; 1 mM DTT, 1 mM Na3VO4; 10 mM NaF), supplemented with mini protease inhibitor cocktail tablet (Roche, Germany). Lysates were centrifuged at 1000 × g, for 5 min at 10 °C and the supernatant collected and centrifuged at 3300 × g, for 5 min at 4 °C. Protein concentration was determined by the Bradford assay (Biorad) and denatured with SDS buffer (0.5 M Tris, 30% glycerol, 10% SDS, 0.6 M DTT, 0.012% bromophenol blue). After heating for 5 min at 95 °C, the samples were frozen at -20 °C until use.

Marques A.P., Cunha-Santos J., Leal H., Sousa-Ferreira L., Pereira de Almeida L., Cavadas C, & Rosmaninho-Salgado J. (2018). Dipeptidyl peptidase IV (DPP-IV) inhibition prevents fibrosis in adipose tissue of obese mice. Biochimica et biophysica acta. General subjects, 1862(3).

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Bcl-2 and Bax Protein Extraction from Pancreas

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The Bcl-2 and Bax proteins from the pancreas tissue were extracted according to the instructions for the protein extraction. After the pancreas was centrifuged, the supernatant was extracted and centrifuged at 4 °C. Protein concentrations were measured by using a Bio-Rad Bradford assay (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Before being transferred to a nitrocellulose membrane, Bcl-2 and Bax protein were separated on 12% SDS–polyacrylamide gel electrophoresis. Then the membranes were blocked with 5% non-fat milk for 2 h at room temperature, followed by incubation with primary antibody (anti-Bcl-2 at 1:500; anti-Bax at 1:800) at 4 °C overnight and secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. In the end, the membranes were treated with emitter-coupled logic and the signals were detected by exposure of the membranes to X-ray films, β-actin was used for normalization. The relative signal intensity was quantified by densitometry with Quantity One software (Bio-Rad Laboratories, Inc).

Fang Y., Zhang Q., Tan J., Li L., An X, & Lei P. (2014). Intermittent hypoxia-induced rat pancreatic β-cell apoptosis and protective effects of antioxidant intervention. Nutrition & Diabetes, 4(9), e131-.

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