Standard dc assay

Manufactured by Bio-Rad

Sourced in United Kingdom

The Standard DC assay is a colorimetric-based protein assay used for the determination of protein concentration. It utilizes a copper-based reagent to react with proteins, producing a blue-purple color that is proportional to the protein present. This assay is compatible with a wide range of samples and can be used with a variety of spectrophotometric methods for quantification.

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Lab products found in correlation

Odyssey blocking buffer, by LI COR (2 mentions) Odyssey blocking buffer, by Cell Signaling Technology (2 mentions) Anti actin, by Cell Signaling Technology (2 mentions) Bis tris gel, by Thermo Fisher Scientific (2 mentions) Odyssey infrared scanner and software, by LI COR (2 mentions) Anti phospho1 recombinant fab, by Bio-Rad (2 mentions) Anti tnap, by R&D Systems (2 mentions) Thermo icapq icp ms, by Thermo Fisher Scientific (1 mentions) Trace nitric acid, by Thermo Fisher Scientific (1 mentions) Equine cytochrome c, by Merck Group (1 mentions) Complete protease inhibitor cocktail, by Bio-Rad (1 mentions) Hrp conjugated secondary antibody, by Merck Group (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions)

Close spelling variants of this product

DC assay (217 citations) Standard DC protein assay (3 citations)

4 protocols using standard dc assay

Quantification of Protein Metal Loading

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The purified MbnH protein concentration was determined using a standard DC assay (Bio-Rad) with equine cytochrome c (Sigma) as the standard curve protein. Metal loading was determined using ICP-MS at the Quantitative Bioelement Imaging Center at Northwestern University. Quantification of Fe was performed after digesting samples in concentrated trace nitric acid (> 69%, Thermo Fisher Scientific) and heating at 65 °C overnight for ~14 h. MilliQ H₂O (18.2 MΩ·cm) was then added to produce a final solution of 3.0% nitric acid (v/v) in a 5 mL sample. A 100 μg/mL Fe element standard (Inorganic Ventures) was used to produce a 100 ng/mL 50 mL quantitative standard in 3.0% nitric acid (v/v). Samples were measured using a Thermo iCapQ ICP-MS (Thermo Fisher Scientific) controlled by the QTEGRA software in the same manner as described previously.^{23 (link)} Quantitative heme loading was achieved in the purified protein as determined by comparison of the Fe content to the protein concentration.

Manesis A.C., Slater J.W., Cantave K., Bollinger JM J.r., Krebs C, & Rosenzweig A.C. (2023). Capturing a bis-Fe(IV) State in Methylosinus trichosporium OB3b MbnH. Biochemistry, 62(5), 1082-1092.

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Western Blot Analysis of Phospho1 and TNAP

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Cells were lysed in RIPA buffer (20mM Tris-HCL, pH. 8.0, 135mM NaCL, 10% glycerol, 1% IGEPAL, 0.1% SDS, 0.5% Na deoxycholate, 2mM EDTA; Invitrogen) containing “complete” protease inhibitor cocktail according to manufacturer’s instructions (Roche) and protein concentration determined using the standard DC assay (Bio-Rad, Hemel Hempsted, UK). Proteins (8 μg) were run in a 10% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences, Nebraska, USA) for 1 hour and probed overnight at 4°C with anti-PHOSPHO1 (recombinant Fab, AbD Serotec, Martinsried/Planegg, Germany) anti-TNAP (R&D, Abingdon, UK) and anti ß-actin (Cell signalling technology, Hitchin, UK) antibodies diluted 1:1000, 1:500 and 1:1000 respectively in Odyssey blocking buffer. After washing in PBS the membranes were incubated with goat anti-Human (800CW), goat anti-Rat (800CW) and goat anti-Rabbit (680RD) for 50 minutes (LI-COR Biosciences, 1:1250 dilution in Odyssey blocking buffer). The resulting blots were subsequently washed in PBS and visualised using the LI-COR Odyssey infra-red scanner and software (LI-COR biosciences) with a scan resolution of 169μm.

Huesa C., Houston D., Kiffer-Moreira T., Yadav M.C., Luis Millan J, & Farquharson C. (2015). The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.. Biochemistry and Biophysics Reports, 4, 196-201.

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Western Blot Analysis of EGF Signaling

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Cells were seeded in 10% FBS overnight to allow for adherence and then serum starved for 24 hr and treated with or without EGF at 100 ng/mL for 10 min at 37°C. Cell lysates were collected on ice with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 5 mM EDTA) supplemented with protease (Roche) and phosphatase (Sigma) inhibitors. Protein concentration was determined using a standard DC assay (Bio-Rad) and equal amounts were loaded and separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with buffer containing 5% BSA in tris-buffered saline with 0.1% Tween-20 (TTBS) for 1 h and incubated overnight with primary antibodies at 4°C. Membranes were washed with TTBS, and then incubated with HRP-conjugated secondary antibodies (Sigma-Aldrich) and developed with an enhanced chemiluminescence detection kit (Pierce). ImageJ software was used to quantify band signal intensity normalized to total protein.

Binder Z.A., Thorne A.H., Bakas S., Wileyto E.P., Bilello M., Akbari H., Rathore S., Ha S.M., Zhang L., Ferguson C.J., Dahiya S., Bi W.L., Reardon D.A., Idbaih A., Felsberg J., Hentschel B., Weller M., Bagley S.J., Morrissette J.J., Nasrallah M.P., Ma J., Zanca C., Scott A.M., Orellana L., Davatzikos C., Furnari F.B, & O’Rourke D.M. (2018). Epidermal Growth Factor Receptor Extracellular Domain Mutations in Glioblastoma Present Opportunities for Clinical Imaging and Therapeutic Development. Cancer cell, 34(1), 163-177.e7.

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Western Blot Analysis of Bone Markers

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Cells were lysed in RIPA buffer (20 mM Tris–HCl, pH. 8.0, 135 mM NaCL, 10% glycerol, 1% IGEPAL, 0.1% SDS, 0.5% Na deoxycholate, 2 mM EDTA; Invitrogen) containing “complete” protease inhibitor cocktail according to manufacturer's instructions (Roche) and protein concentration determined using the standard DC assay (Bio-Rad, Hemel Hempsted, UK). Proteins (8 µg) were run in a 10% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences, Nebraska, USA) for 1 h and probed overnight at 4 °C with anti-PHOSPHO1 (recombinant Fab, AbD Serotec, Martinsried/Planegg, Germany) anti-TNAP (R&D, Abingdon, UK) and anti ß-actin (Cell signaling technology, Hitchin, UK) antibodies diluted 1:1000,1:500 and 1:1000 respectively in Odyssey blocking buffer. After washing in PBS the membranes were incubated with goat anti-Human (800CW), goat anti-Rat (800CW) and goat anti-Rabbit (680 RD) for 50 min (LI-COR Biosciences, 1:1250 dilution in Odyssey blocking buffer). The resulting blots were subsequently washed in PBS and visualized using the LI-COR Odyssey infrared scanner and software (LI-COR biosciences) with a scan resolution of 169 µm.

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