Coomassie g 250 stain

Manufactured by Bio-Rad

Coomassie G-250 stain is a colorimetric assay used for quantitative protein determination. It binds to proteins in a non-specific manner, resulting in a color change that can be measured spectrophotometrically.

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6 protocols using coomassie g 250 stain

Characterization of LPMO Enzymes by SDS-PAGE and Spectroscopy

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Culture supernatant and concentrated enzyme were analyzed using 12% SDS-PAGE gels to confirm the presence of the LPMO enzyme. Protein bands were visualized by staining with colloidal Coomassie G-250 stain (Bio-Rad) and the PageRuler prestained protein ladder (Thermo Fisher Scientific) was used for mass determination.
A dilution series of bovine serum albumin (0.3–0.05 mg/L) was added to each gel to get an estimation of the protein concentration of the ultrafiltrated culture supernatant, using the digital imaging software ImageJ as described earlier [25 (link),31 ].
The concentration of purified enzyme was measured using a Nanodrop device with extinction coefficients of 54360 M^-1.cm^-1, 52870 M^-1.cm^-1 and 52870 M^-1.cm^-1 for HjLPMO9A wildtype, variant Y24A and variant Y211A, respectively.

Danneels B., Tanghe M., Joosten H.J., Gundinger T., Spadiut O., Stals I, & Desmet T. (2017). A quantitative indicator diagram for lytic polysaccharide monooxygenases reveals the role of aromatic surface residues in HjLPMO9A regioselectivity. PLoS ONE, 12(5), e0178446.

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GST Pull-down Protein Visualization

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GST Pull-down samples were run on a 10% SDS-PAGE gel at 150 volts for 1.5 hours. The gel was washed in water for 5 minutes and repeated 3 more times. The gel was incubated with 50 ml Coomassie G-250 Stain (Bio-Rad) in an orbital shaker for 1 hour. The gel was rinsed with water for 30 minutes.

Ben-Menachem R., Wang K., Marcu O., Yu Z., Lim T.K., Lin Q., Schueler- Furman O, & Pines O. (2018). Yeast aconitase mitochondrial import is modulated by interactions of its C and N terminal domains and Ssa1/2 (Hsp70). Scientific Reports, 8, 5903.

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Phosphoprotein Gel Staining Protocol

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To detect overall phosphorylation changes, a 15% SDS-PAGE gel was stained with Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen, P33300, Carlsbad, CA) and destained with Pro-Q Diamond Phosphoprotein Gel Distaining Solution (Invitrogen, P33310, Carlsbad, CA). The gel was then stained with Coomassie G-250 Stain (Bio-Rad Inc., 1,610,786, Hercules, CA) to determine total protein levels. Gel images were captured with ChemiDoc MP (Bio-Rad, Inc., 13,036, Hercules, CA) and band densities were determined and analyzed by using ImageLab 6.0.1 software and Microsoft Excel.

Capote A.E., Batra A., Warren C.M., Chowdhury S.A., Wolska B.M., Solaro R.J, & Rosas P.C. (2021). B-arrestin-2 Signaling Is Important to Preserve Cardiac Function During Aging. Frontiers in Physiology, 12, 696852.

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Cytokine Size and Mobility Characterization

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Cytokine hydrodynamic size was measured by dynamic light scattering (Wyatt DynaPro Plate Reader III) using 2–4 averages of 10–30 s acquisitions. Electrophoretic mobility was measured via polyacrylamide gel electrophoresis under reducing conditions (50 mM dithiothreitol, Bio-Rad). Protein bands were visualized with a Coomassie G250 stain (Bio-Rad) and imaged using a Licor CLx gel imager.

Perdue L.A., Do P., David C., Chyong A., Kellner A.V., Ruggieri A., Kim H.R., Salaita K., Lesinski G.B., Porter C.C, & Dreaden E.C. (2020). Optical Control of Cytokine Signaling via Bioinspired, Polymer-Induced Latency. Biomacromolecules, 21(7), 2635-2644.

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Gel Staining and Membrane Visualization

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Where indicated, gels after native PAGE or SDS-PAGE were washed with deionized water three times for 5 min and incubated with Coomassie G-250 stain (Bio-Rad) for 1 h. The gels were washed with water after to remove the excess of the dye and imaged. Where indicated, membranes after protein transfer were incubated with Ponceau S solution (Sigma) for 10 min, then were washed with water to remove the excess of the dye and imaged.

McNutt S.W., Roychowdhury T., Pasala C., Nguyen H.T., Thornton D.T., Sharma S., Botticelli L., Digwal C.S., Joshi S., Yang N., Panchal P., Chakrabarty S., Bay S., Markov V., Kwong C., Lisanti J., Chung S.Y., Ginsberg S.D., Yan P., DeStanchina E., Corben A., Modi S., Alpaugh M., Colombo G., Erdjument-Bromage H., Neubert T.A., Chalkley R.J., Baker P.R., Burlingame A.L., Rodina A., Chiosis G, & Chu F. (2024). Phosphorylation-Driven Epichaperome Assembly: A Critical Regulator of Cellular Adaptability and Proliferation. Research Square.

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Mitochondria Isolation and Enzyme Assay

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Isoelectric pH gradient (IPG) strips (pH 3.0–10.0; NL, 17 cm), urea, pharmalyte (pH 3–10), glycerol (87% w/w), Tris (electrophoresis grade), 1,2-di(dimethylamino)ethane (TEMED; electrophoresis purity reagent), acrylamide (40% solution; acrylamide-to-bisacrylamide ratio, 37.5:1), 3-[(3-cholamidoprpyl) dimethylammonio]-1-propanesulfonate (CHAPS; electrophoresis grade), thiourea (ACS grade), dithiothreitol (DTT, electrophoresis grade), iodoacetamide (electrophoresis grade), mineral oil, Coomassie G-250 stain and low-melting-point agarose were obtained from Bio-Rad. Animal cell/tissue quality purified mitochondria isolation kits were purchased from Genmed Scientifics, and the enzyme activity assay kits were purchased from Nanjing Jiancheng Biotechnology Institution. High-purity water prepared from the Milli-Q gradient water purification system (Millipore) was used for all the experiments in this study.

Peng M., Han J., Li L, & Ma H. (2016). Suppression of fat deposition in broiler chickens by (-)-hydroxycitric acid supplementation: A proteomics perspective. Scientific Reports, 6, 32580.

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