Bradford protein assay system

Manufactured by Bio-Rad

Sourced in United States, United Kingdom

The Bradford protein assay system is a colorimetric analytical procedure used to measure the concentration of protein in a solution. It utilizes the principle of protein-dye binding to quantify the amount of protein present in the sample.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (6 mentions) Horseradish peroxidase, by Merck Group (3 mentions) Hybond, by Cytiva (3 mentions) E cadherin, by Abcam (2 mentions) N cadherin, by Abcam (2 mentions) Interleukin 6 (il 6), by Bioworld Technology (2 mentions) Enhanced chemiluminescence western blot detection system, by Cytiva (2 mentions) Antibodies for ing5 and smad3, by Proteintech (2 mentions) Enhanced chemiluminescence western blotting detection system, by Cytiva (2 mentions) Ing5 antibody, by Proteintech (2 mentions) Enhanced chemiluminescence reagent, by Cytiva (2 mentions) Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (2 mentions) Secondary antibody conjugated with horseradish peroxidase, by Merck Group (1 mentions) Ceacam6, by Abcam (1 mentions) Odyssey blocking buffer, by LI COR (1 mentions) Halt phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Iblot device, by Thermo Fisher Scientific (1 mentions) Parp1, by Bio-Techne (1 mentions) Bis tris gel, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Bradford assay (3878 citations) Bio-Rad protein assay (2654 citations) Bradford protein assay (1622 citations) Bio-Rad protein assay system (60 citations) Bradford assay system (2 citations)

15 protocols using bradford protein assay system

Western Blot Analysis of Protein Expression

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Cells were lysed in lysis buffer and protein concentrations of the lysates were determined using the Bradford protein assay system (Bio‐Rad, Hercules, CA, USA). Equal amounts (30μg protein each lane) of total cellular protein were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The membrane was blocked and then incubated with primary antibody overnight at 4°C. Primary antibodies used included antibodies for ING5 and SMAD3 (Proteintech Group, Inc., Rosemont, IL, USA); IL‐6 (Bioworld Technology Co., Ltd., Nanjing, China); pAKT (Ser473/Thr308) and STAT3 (Cell Signaling
Technology, Danvers, MA, USA); p‐STAT3 (Y705), AKT, β‐catenin, p‐β‐catenin (Ser33/S37), E‐cadherin, N‐cadherin, Snail, Slug, Twist, EGFR, and CEACAM6 (Abcam, Cambridge, MA, USA); and β‐actin (Actin) (Sigma‐Aldrich, St. Louis, MO, USA). The membrane was incubated with corresponding secondary antibody conjugated with horseradish peroxidase (1:5000, Sigma‐Aldrich) at room temperature for one hour. The blots were developed using an enhanced chemiluminescence Western blot detection system (Amersham Bioscience, Buckinghamshire, UK).

Liu X., Meng J., Zhang X., Liang X., Zhang F., Zhao G, & Zhang T. (2019). ING5 inhibits lung cancer invasion and epithelial–mesenchymal transition by inhibiting the WNT/β‐catenin pathway. Thoracic Cancer, 10(4), 848-855.

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Western Blot Analysis of Cerebellar Proteins

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Mouse cerebellar tissues and cell pellets were homogenized with a cell lysis buffer (Cell Signaling) with Halt phosphatase inhibitor (Thermo-Fisher), complete protease inhibitors (Roche) and PSMF (Sigma-Aldrich). The supernatant was recovered by centrifugation (16,000g, 15mins). Protein concentration was assayed by the Bradford protein assay system (Bio-rad). Twenty micrograms of lysates were loaded into 4–12% Bis—Tris gels (Invitro-gen). Electrophoresis was carried out according to the manufacturer’s recommendations. Following electrophoresis, the proteins were transferred to nitrocellulose membranes by the iBlot device (Invitrogen), blocked with an Odyssey blocking buffer (LI-COR Biotechnology) for 1 h. Membranes were incubated overnight with the following primary antibodies in blocking buffer: Iba1(Wako), CD11b(Abcam), MUTYH(Abcam), PARP-1(Trevigen), Frataxin (Santa Cruz), Actin(Abcam), AT1(Abcam), Tubulin (Abcam). Subsequently, the membranes were incubated with a corresponding pair of IRDye 680CW and IRDye 800CW-coupled secondary antibodies (LI-COR). Proteins were visualized with the Odyssey infrared imager and software (LI-COR) according the manufacturer’s instruction.

Shen Y., McMackin M.Z., Shan Y., Raetz A., David S, & Cortopassi G. (2016). Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34. PLoS ONE, 11(3), e0151026.

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Immunoprecipitation Protocol for Protein Complexes

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Cells were grown to confluence in 10 cm plates. Plates were washed twice on ice in ice-cold PBS and then lysis buffer was added with a protease inhibitor cocktail (Roche). Lysates collected after 10 min and pre-cleared by centrifugation at 13,000 G for 10 min at 4 °C. The supernatant was removed from the debris pellet and transferred to a clean tube. Protein concentration was calculated using the Bradford Protein Assay system (Bio-Rad). Fifty microlitres of Dynabeads (Thermo) were prepared according to manufacturer's instructions. Antibodies (2–4 μg) were bound to the beads by incubation at room temperature with rotation for 30 min. Species matched IgG (Rabbit IgG sc-2027, Mouse IgG sc-2025, Santa Cruz) were bound to beads as a control. Two milligram of protein lysate was added to beads and incubated overnight at 4 °C with rotation. The following day, beads were washed three times for 5 min on ice in 700 μl NP-40 wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA and 1% NP40) containing protease and phosphatase inhibitors (Roche) followed by separation on a magnet. Beads were separated from antibody–protein complexes by boiling for 5 min in 2 × Laemelli buffer and the eluate separated by a magnet loaded onto a gel for SDS–PAGE electrophoresis. The lysate was run alongside as an input for the immunoprecipitation.

Maruthappu T., Chikh A., Fell B., Delaney P.J., Brooke M.A., Levet C., Moncada-Pazos A., Ishida-Yamamoto A., Blaydon D., Waseem A., Leigh I.M., Freeman M, & Kelsell D.P. (2017). Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16. Nature Communications, 8, 14174.

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Cell Lysis and Protein Quantification

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RIPA buffer containing protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) was used to lyse the cells (1 × 10⁶ cells/25 μL lysis buffer). The process of the lysis was carried out for 60 min on ice at 4 °C The Bradford protein assay system (Bio-Rad Laboratories, Hemel Hempstead, UK) was used to determine the concentration of the protein.

Świerczewska M., Sterzyńska K., Wojtowicz K., Kaźmierczak D., Iżycki D., Nowicki M., Zabel M, & Januchowski R. (2019). PTPRK Expression Is Downregulated in Drug Resistant Ovarian Cancer Cell Lines, and Especially in ALDH1A1 Positive CSCs-Like Populations. International Journal of Molecular Sciences, 20(8), 2053.

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Western Blot Analysis of Nuclear and Cytosolic Proteins

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Cells were lysed in lysis buffer containing 150mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 50mM Tris (pH 8.0), and 1:25 protease inhibitor cocktail for total protein. Nuclear and cytosolic protein were extracted using Nuclear/cytosol fractionation kit (BioVision Incorporated, CA). Protein concentrations of the lysates were determined by the Bradford protein assay system (Bio-Rad, Hercules, CA). Equal amounts of protein (30μg protein each lane) were separated by SDS-PAGE, transferred to nitrocellulose membranes (Hybond C, Amersham, UK). Immunoblots were blocked with 5% skim milk in TBS/Tween 20 (0.05%, v/v) for 1 hour at RT. The membrane was incubated with primary antibody overnight at 4°C. Primary antibodies used include ING5 antibody (Proteintech Group, Inc. IL), H2AX antibody (Santa Cruz Biotechnology Inc.), antibodies for E-cadherin, Snail and N-cadherin (Abcam) and β-actin (Actin) antibody (Sigma). The membrane was incubated with corresponding secondary antibody conjugated with horseradish peroxidase (Sigma) (1:5000) at RT for 1h. The blots were developed using an enhanced chemiluminescence western blotting detection system (Amersham Bioscience, UK).

Zhang F., Zhang X., Meng J., Zhao Y., Liu X., Liu Y., Wang Y., Li Y., Sun Y., Wang Z., Mei Q, & Zhang T. (2015). ING5 inhibits cancer aggressiveness via preventing EMT and is a potential prognostic biomarker for lung cancer. Oncotarget, 6(18), 16239-16252.

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Western Blot Analysis of Protein Expression

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Cells were lysed in lysis buffer containing 150 mM NaCl, 1%NP40, 0.5%deoxycholic acid, 0.1%SDS, 50 mMTris (pH 8.0), and 1:25 protease inhibitor cocktail. Protein concentrations of the lysates were determined by the Bradford protein assay system (Bio-Rad, Hercules, CA). Equal amounts of protein (30 μg protein each lane) were separated by SDS-PAGE, transferred to nitrocellulose membranes (Hybond C, Amersham, UK). Immunoblots were blocked with 5% skim milk in TBS/Tween20 (0.05%, v/v) for 1 hour at RT. The membrane was incubated with primary antibody overnight at 4°C. Primary antibodies used include ING5 antibody (Proteintech Group, Inc. IL), p21 antibody (Santa Cruz Biotechnology Inc.), antibodies for Ac-H3K18, Ac-H4K16, p53, Ac-p53K382, Bax (Abcam), β-actin (Actin) antibody (Sigma), and pan-Acetyl antibody (Jingjie PTM Biolab). The membrane was incubated with corresponding secondary antibody conjugated with horseradish peroxidase (Sigma) (1:5000) at RT for 1h. The blots were developed using an enhanced chemiluminescence western blotting detection system (Amersham Bioscience, UK).

Zhang T., Meng J., Liu X., Zhang X., Peng X., Cheng Z, & Zhang F. (2017). ING5 differentially regulates protein lysine acetylation and promotes p300 autoacetylation. Oncotarget, 9(2), 1617-1629.

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Isolation and Quantification of Cellular and Secreted Proteins from Ovarian Cancer Cell Lines

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Whole cell lysates from drug-sensitive and drug-resistant ovarian cancer cells (1 × 10⁶ cells/20 μL lysis buffer) were lysed using containing protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min on ice. The lysates were centrifuged at 8000× g for 10 min at 4 °C, and protein concentration was quantified using the Bradford protein assay system (Bio-Rad Laboratories, Hemel Hempstead, UK). The isolation of proteins from media was preceded by culturing cells in serum-free media for 72 h. After that, the media was centrifuged at 15,000 rpm for 30 min at RT. Then, the supernatants were transferred to Amicon Ultra-15 3K centrifuge filter devices and centrifuged according the manufacturer’s protocols (40 min, 4000× g, RT, swinging-bucket rotor).

Sterzyńska K., Klejewski A., Wojtowicz K., Świerczewska M., Andrzejewska M., Rusek D., Sobkowski M., Kędzia W., Brązert J., Nowicki M, & Januchowski R. (2018). The Role of Matrix Gla Protein (MGP) Expression in Paclitaxel and Topotecan Resistant Ovarian Cancer Cell Lines. International Journal of Molecular Sciences, 19(10), 2901.

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Protein Isolation and Glycosidase Treatment

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The cells (1 × 10⁶ cells/25 μL lysis buffer) were lysed in RIPA buffer containing protease inhibitor cocktail (ROCHE) for 60 min on ice at 4°C. The lysates were centrifuged at 12000 × g for 15 min at 4°C, and protein concentrations were determined using the BioRad (Hercules, CA) Bradford protein assay system. To isolate proteins from media, cells were cultured in serum-free media for 72 hours. Next, the media was centrifuged at 15 000 rpm for 30 min at RT. Then, the supernatants were transferred to Amicon Ultra-15 3K centrifuge filter devices and centrifuged according to the manufacturer’s instructions (60 min, 4 000 x g, RT, swinging-bucket rotor). Glycosidase treatment was performed by incubating 35 μg of cell lysate with 5 μl of PNGase F, 0.5 μl of β-ME and 1 μl of SDS at 37°C for 10 min.

Klejewski A., Sterzyńska K., Wojtowicz K., Świerczewska M., Partyka M., Brązert M., Nowicki M., Zabel M, & Januchowski R. (2017). The significance of lumican expression in ovarian cancer drug-resistant cell lines. Oncotarget, 8(43), 74466-74478.

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Protein to DNA Content Ratio

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To evaluate changes in cell size, the protein to DNA content ratio was determined. Total cellular protein was measured using the Bradford Protein Assay system (Bio-Rad Laboratories, Hercules, CA, USA). Genomic DNA was isolated using a phenol chloroform isopropanol extraction and the DNA concentration was measured using a spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA). The protein to DNA content ratio was calculated from the CPO colons and compared to the region-matched controls.

Do Y.S., Myung S.J., Kwak S.Y., Cho S., Lee E., Song M.J., Yu C.S., Yoon Y.S, & Lee H.K. (2015). Molecular and Cellular Characteristics of the Colonic Pseudo-obstruction in Patients With Intractable Constipation. Journal of Neurogastroenterology and Motility, 21(4), 560-570.

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Immune Modulation by hUCMSCs

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2×10⁵ hUCMSCs or 1.5×10⁵ De-hUCMSCs were seeded in one well of the six-well plates. After 24 hours, cells were rinsed with PBS and 1 ml serum-free α-MEM medium (Thermofisher, Waltham, MA, USA) was added. Medium was collected 48 h later and used immediately or stored at −80°C. Colons were homogenized in PBS with 0.5% 100x Triton (Sigma, St. Louis, MO, USA) and protease inhibitor cocktail. Lysates were incubated at 4°C for 30 mins, followed by 14,000 rpm centrifuge at 4°C. Supernatant was collected and protein concentration was measured by the Bradford protein assay system (Bio-Rad). The enzyme-linked immunosorbent assay (ELISA) kits used were Mouse IL-6, IL-10 ELISA Kits (ThermoFisher Scientific, Waltham, MA, USA; EM2IL6, EM2IL10, EMTNFA), Mouse IL-17a ELISA kit (Invitrogen, Carlsbad, CA, USA; KMC3021), Prostaglandin E₂ EIA Kit-Monoclonal (Cayman, Ann Arbor, MI, USA; 514010) and Human IL-2, IL-10 (ThermoFisher Scientific; EH2IL2, EHIL10).

Yang F.Y., Chen R., Zhang X., Huang B., Tsang L.L., Li X, & Jiang X. (2018). Preconditioning Enhances the Therapeutic Effects of Mesenchymal Stem Cells on Colitis Through PGE2-Mediated T-Cell Modulation. Cell Transplantation, 27(9), 1352-1367.

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