Protein assay dye reagent

Manufactured by Bio-Rad

Sourced in United States, Germany, Italy, Canada, Switzerland, Denmark

Protein Assay Dye Reagent is a colorimetric solution used for the quantification of protein concentration in aqueous samples. The reagent binds to proteins, resulting in a color change that can be measured using a spectrophotometer. This allows for the determination of protein levels in a variety of biological and chemical samples.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (22 mentions) Protease inhibitor cocktail, by Merck Group (18 mentions) Complete protease inhibitor cocktail, by Roche (17 mentions) Nitrocellulose membrane, by Bio-Rad (13 mentions) Pvdf membrane, by Bio-Rad (8 mentions) Ripa buffer, by Thermo Fisher Scientific (8 mentions) Chemidoc mp imaging system, by Bio-Rad (7 mentions) Nitrocellulose membrane, by Cytiva (7 mentions) Pvdf membrane, by Merck Group (7 mentions) Spark multimode microplate reader, by Tecan (7 mentions) β actin, by Cell Signaling Technology (7 mentions) Chemidoc imaging system, by Bio-Rad (6 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (6 mentions) Enhanced chemi luminescence (ecl), by GE Healthcare (6 mentions) β actin, by Merck Group (6 mentions) Odyssey infrared imaging system, by LI COR (5 mentions) Protease inhibitor, by Roche (5 mentions) Gapdh, by Cell Signaling Technology (5 mentions) Immobilon p membrane, by Merck Group (5 mentions) Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (5 mentions)

Close spelling variants of this product

Bradford assay (Protein Assay Dye Reagent (2 citations) Protein Assay Dye Reagent Concentrate #500-0006 (2 citations) Protein Assay concentrated dye reagent (2 citations) Protein Assay Dye Reagent Concentration (5 citations) Protein Assay Dye Reagent Concentrate (408 citations) Bradford protein assay dye reagent (18 citations) Protein Assay Dye Reagent Concentrate Kit (5 citations) Coomassie Protein Assay Dye Reagent Concentrate (2 citations) Protein Assay Dye Reagent kit (5 citations) Dye reagent for protein assay (2 citations)

425 protocols using protein assay dye reagent

Quantifying Biomolecules in Samples

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Protein contents were measured according to the method of Bradford [16 (link)] using the Bio-Rad protein assay dye reagent diluted with five volumes of deionized water. The absorbances at 595 nm were measured after 10-μL samples (1 mg/mL) and 200-μL diluted Bio-Rad protein assay dye reagent were thoroughly mixed and kept for 10 minutes. Bovine serum albumin (0–500 μg/mL) was used as the standard.
The total polysaccharide contents were determined following the method of Chaplin and Kennedy [17 ]. A 200-μL sample, 1-mL concentrated sulfuric acid, and 200-μL phenol (5%) were mixed and kept at room temperature for 10 minutes. The absorbance of the samples at 490 nm was measured after keeping at room temperature for an additional 30 minutes. Glucose (0–100 μg/mL) was used as the standard to establish a calibration curve.
The concentration of total phenolic compounds was determined spectrophotometrically using Folin–Ciocalteu’s reagent and the method of Quettier-Deleu et al [18 ]. The absorbance of the samples at 760 nm was determined after mixing 0.2-mL sample, 1-mL Folin–Ciocalteu’s phenol reagent, 0.8-mL sodium carbonate (7.5%), and incubated for 30 minutes. Gallic acid (0–100 μg/mL) was used as the standard for a calibration curve and the results were expressed as μg of gallic acid equivalents per mg of sample (μg GAE/mg).

Chen P.H., Weng Y.M., Yu Z.R., Koo M, & Wang B.J. (2016). Extraction temperature affects the activities of antioxidation, carbohydrate-digestion enzymes, and angiotensin-converting enzyme of Pleurotus citrinopileatus extract. Journal of Food and Drug Analysis, 24(3), 548-555.

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Protein Extraction and Western Blotting Protocol

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Cells were lysed in RIPA lysis buffer (New England Biolabs), sonicated and protein concentrations were measured using the Protein Assay Dye Reagent (Biorad). Samples were mixed with NuPAGE LDS Sample Buffer (Invitrogen), boiled for 5 minutes at 98°C and proteins were separated on SDS-PAGE gels and transferred onto Amersham™ Protran nitrocellulose membranes (0.45μm, Cytiva) for confirmation of the knockout status of cell lines, or onto Amersham™ Hybond PVDF membrane (0.2 μm, Cytiva) for the identification of Pt-alkyne-53 interactors. After 1 hour of blocking in 5% milk in TBS-T (0.1% Tween 20 in 1x Tris-buffered saline), membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies used were against FANCD2 (diluted 1:1,000, EPR2302 Abcam), XPF (diluted 1:500, 3F2/3 Santa Cruz), PCNA (diluted 1:500, PC10 Santa Cruz) and H3 (diluted 1:15000, ab1791 Abcam), and as a loading control, against Tubulin (diluted 1:10,000, DM1A Cell Signaling). Anti-mouse and anti-rabbit HRP-conjugated goat secondary antibodies (Jackson Immunochemicals) were used at a final dilution of 1:5,000. Immunoblots were imaged using a Curix 60 (AGFA) table-top processor.

Moretton A., Slyskova J., Simaan M.E., Arasa-Verge E.A., Meyenberg M., Cerrón-Infantes D.A., Unterlass M.M, & Loizou J.I. (2022). Clickable Cisplatin Derivatives as Versatile Tools to Probe the DNA Damage Response to Chemotherapy. Frontiers in Oncology, 12, 874201.

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Protein Extraction and Immunoblotting from Mouse Ileum

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Flash-frozen mouse ileum was resuspended in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 5 mM EDTA, 0.1% SDS), containing protease inhibitors (Roche, cat. #: 11836170001, South San Francisco, CA, USA). Tissue was homogenized and then centrifuged at 12,000× g for 15 min, after which supernatants were collected. Protein concentration was determined using the Protein Assay Dye Reagent (Bio-Rad, cat. #: 500-0006, Hercules, CA, USA). 40 µg protein mixed with 4× Laemmli sample buffer were incubated at 37 °C for 15 min and subjected to SDS-PAGE. After overnight transfer onto PVDF membrane (Bio-Rad, cat. #: 1620177, Hercules, CA, USA), membrane was incubated overnight with primary antibody at 4 °C. Membrane was washed and then incubated with secondary antibody for 1 h, followed by application of chemiluminescent reagent ECL (GE Healthcare, cat. #: RPN2232, Chicago, IL, USA) and imaging on the ChemiDoc Imaging System (Bio-Rad), after which relative protein concentration was determined by densitometry. The primary antibodies used were DUOX2 (Santa Cruz, cat. #: sc-398681, Dallas, TX, USA), GAPDH (Santa Cruz, cat. #: sc-32233, Dallas, TX, USA). The secondary antibody was mouse IgG kappa binding protein conjugated to horseradish peroxidase (Santa Cruz, cat. #: sc-516102, Dallas, TX, USA).

Lai K.K., Hu X., Chosa K., Nguyen C., Lin D.P., Lai K.K., Kato N., Higuchi Y., Highlander S.K., Melendez E., Eriguchi Y., Fueger P.T., Ouellette A.J., Chimge N.O., Ono M, & Kahn M. (2021). p300 Serine 89: A Critical Signaling Integrator and Its Effects on Intestinal Homeostasis and Repair. Cancers, 13(6), 1288.

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Nuclear Extract Preparation from S2 Cells

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S2 cells were harvested, washed in phosphate-buffered saline (PBS) and resuspended in three volumes of low salt buffer (10 mm Hepes pH 7.6, 1.5 mm MgCl₂, 10 mm KCl, 1.0 mm dithiothreitol (DTT)). After incubation on ice for 10 min, cells were collected by centrifugation at 21 100 × g for 1 min at 4°C. The supernatant was discarded, and nuclei were resuspended in 1.5 volumes of high salt buffer (20 mm Hepes pH 7.6, 1.5 mm MgCl₂, 420 mm NaCl, 0.2 mm ethylenediaminetetraacetic acid (EDTA), 20% (v/v) glycerol, 1.0 mm DTT). The suspension was incubated for 20 min on ice and subsequently centrifuged at 21 100 × g for 30 min at 4°C. The supernatant (nuclear extract) was aliquoted, frozen in liquid nitrogen and stored at −80°C.
Preparation of nuclear extract from Drosophila embryos was done as described previously (16 (link)).
The protein concentration of nuclear extracts was determined using Protein Assay Dye Reagent (Bio-Rad) according to the manufacturer's instructions using BSA (Roth) as a standard.

Mačinković I., Theofel I., Hundertmark T., Kovač K., Awe S., Lenz J., Forné I., Lamp B., Nist A., Imhof A., Stiewe T., Renkawitz-Pohl R., Rathke C, & Brehm A. (2019). Distinct CoREST complexes act in a cell-type-specific manner. Nucleic Acids Research, 47(22), 11649-11666.

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Protein Extraction and Immunoblotting Protocol

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Cells were lysed using lysis-C buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS and 0.0002% (v/v) bromophenol blue] containing protease inhibitor (Bioman). The cells were homogenized on ice using an ultrasonic homogenizer (LABSONIC M ultrasonic homogenizer) with 60% amplitude and 0.6 cycle duration for 2 min. Cell lysate was centrifuged at 16,000 × g for 30 min at 4 °C. The supernatants were collected and measured protein concentrations with protein assay dye reagent (Bio-Rad Laboratories). Protein extracts were separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore) and immunoblotted with antibodies. The membrane was blocked in 5% non-fat milk/PBST and incubated overnight with primary antibody diluted in blocking buffer at 4 °C: mouse anti-MYCN (abcam; 1:1000), rabbit anti-MTHFD2 (Genetex; 1:1000), rabbit anti-PAICS (Genetex; 1:1000), mouse anti-β-actin (Millipore; 1:5000), and mouse anti-α-tubulin (Genetex; 1:1000). The membrane was then treated with secondary HRP-conjugated antibody anti-rabbit or anti-mouse IgG (Sigma-Aldrich; 1:100,000) for 2 h at room temperature. Images were acquired using ECL substrate (BioRad) and FluorChem M (ProteinSimple).

Cheung C.H., Hsu C.L., Tsuei C.Y., Kuo T.T., Huang C.T., Hsu W.M., Chung Y.H., Wu H.Y., Hsu C.C., Huang H.C, & Juan H.F. (2019). Combinatorial targeting of MTHFD2 and PAICS in purine synthesis as a novel therapeutic strategy. Cell Death & Disease, 10(11), 786.

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Protein Extraction and Western Blot

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We homogenized tissues in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 1 mM Na₃VO₄, 5 mM NaF, 1 mM phenylmethanesulfonylfluoride, 25 mM glycerol 2-phosphate and freshly added protease inhibitor tablet) and then incubated them for 1 h at 4 °C. Cell lysates were produced in an SDS lysis buffer (100 mM Tris pH 7.5, 130 mM NaCl, 1% NP-40, 0.1% SDS, 0.2% sodium deoxycholate, 1 mM Na₃VO₄, 1 mM NaF, 100 mM Na₄P₂O₇, 1 mM phenylmethanesulfonylfluoride, 25 mM glycerol 2-phosphate and freshly added protease inhibitor tablet) and sonicated for three 5 s pulses at an output power of 6. We centrifuged crude lysates at 14,000 g for 15 min twice and determined the protein concentration using Bio-Rad Protein Assay Dye Reagent. Samples were diluted in SDS sample buffer. Bound proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Bio-Rad). Individual proteins were detected with specific antibodies and visualized on film using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and Western Lightning Enhanced Chemiluminescence (Perkin Elmer Life Sciences).

Reilly S.M., Ahmadian M., Zamarron B.F., Chang L., Uhm M., Poirier B., Peng X., Krause D.M., Korytnaya E., Neidert A., Liddle C., Yu R.T., Lumeng C.N., Oral E.A., Downes M., Evans R.M, & Saltiel A.R. (2015). A subcutaneous adipose tissue–liver signalling axis controls hepatic gluconeogenesis. Nature Communications, 6, 6047.

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Protein Extraction and Western Blot Analysis

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Upon harvest, cells were re-suspended with TBSN buffer with protease inhibitors and phosphatase inhibitors and sonicated. After were collected, protein concentrations were measured using Protein Assay Dye Reagent from Bio-Rad. Equal amounts of protein from each sample were mixed with SDS loading buffer and resolved by SDS-PAGE. Upon transferring to PVDF membranes, proteins were probed with indicated antibodies. For IP, cell lysates were incubated with indicated antibodies overnight at 4°C, followed by 1 hr of incubation with protein A/G plus-Agarose beads. After supernatants were removed, beads were washed with high salt and low salt TBSN buffer, and resolved by SDS-PAGE.

Chen L., Li J., Farah E., Sarkar S., Ahmad N., Gupta S., Larner J, & Liu X. (2016). Co-targeting HSP90 and its client proteins for treatment of prostate cancer. Molecular cancer therapeutics, 15(9), 2107-2118.

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Protein Expression and Apoptosis Analysis

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RASMCs were lysed by addition of protein extraction reagent (Pierce) with protease inhibiter cocktail (Sigma). The concentration of protein in cell lysates was determined using protein assay dye reagent (Bio-Rad Laboratories, Hercules, CA). Monoclonal V5-horseradish peroxidase antibody (Thermo Fisher Scientific) was used at 1:5,000 dilution, polyclonal cleaved caspase-3 antibody at 1:100 dilution, and polyclonal caspase-3 antibody (EMD Millipore, Billerica, MA) at 1:1,000 dilution. P27^Kip1 (1:1,000 dilution), GAPDH (1:2,000 dilution), E2F transcription factor 1 (E2F1, 1:300 dilution), and proliferating cell nuclear antigen (PCNA, 1:2,000 dilution) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Growth arrest and DNA damage (GADD45β and GADD45γ) antibodies were obtained from Abcam (Cambridge, MA). Protein bands were visualized with SuperSignal chemiluminescence substrate (Thermo Fisher Scientific).

Liu D., Pattabiraman V., Bacanamwo M, & Anderson L.M. (2016). Iroquois homeobox transcription factor (Irx5) promotes G1/S-phase transition in vascular smooth muscle cells by CDK2-dependent activation. American Journal of Physiology - Cell Physiology, 311(2), C179-C189.

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Insulin Secretion in INS-1E Cells

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Insulin secretion was measured as described (22 (link)). Briefly, INS-1E cells were washed with modified Krebs-Ringer bicarbonate HEPES solution (KRB), incubated for 30 min with KRB without glucose, and insulin secretion was induced by 30 min incubation with KRB containing 1.67 or 16.7 mM glucose with or without 10 μM forskolin. Insulin was measured by ELISA (Mercodia, Uppsala, Sweden) in cell-free supernatants and acid-ethanol extracted cell lysates. Total protein was measured in cell lysates using the Protein Assay Dye Reagent (Biorad).

Abdulkarim B., Nicolino M., Igoillo-Esteve M., Daures M., Romero S., Philippi A., Senée V., Lopes M., Cunha D.A., Harding H.P., Derbois C., Bendelac N., Hattersley A.T., Eizirik D.L., Ron D., Cnop M, & Julier C. (2015). A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly. Diabetes, 64(11), 3951-3962.

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Protein Extraction and Western Blot

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Cell lysates were prepared using Triton X‐100/SDS lysis buffer supplemented with 4% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. Protein concentrations were determined using Bio‐Rad Protein Assay Dye Reagent and made into samples with 30 μg of protein. Samples were run on a 4%‐20% SDS‐PAGE gel and transferred using a Bio‐Rad Semi‐dry Transfer Cell. Membranes were blocked with 5% milk and probed with antibodies described in Materials.

Bunch B.L., Ma Y., Attwood K., Amable L., Luo W., Morrison C., Guru K.A., Woloszynska‐Read A., Hershberger P.A., Trump D.L, & Johnson C.S. (2019). Vitamin D3 enhances the response to cisplatin in bladder cancer through VDR and TAp73 signaling crosstalk. Cancer Medicine, 8(5), 2449-2461.

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