Coomassie blue solution

Manufactured by Merck Group

Sourced in United States, Germany

Coomassie Blue solution is a commonly used dye in molecular biology and biochemistry laboratories. It is a protein stain used to detect and quantify proteins in various applications, such as gel electrophoresis and Western blotting. The solution contains the Coomassie Brilliant Blue dye, which binds to the basic and aromatic amino acid residues in proteins, resulting in a blue color.

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Coomassie Blue G-250 solution Coomassie Blue R-250 solution Coomassie Brilliant Blue Solution Coomassie brilliant blue R solution Coomassie Brilliant Blue R-250 solution Coomassie blue TM-blue

12 protocols using coomassie blue solution

Cytotoxicity and Colony Assays

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Cells were seeded at a density of 5,000 cells per well in 96-well plates and exposed to increasing concentrations of various targeted and cytotoxic agents for 72 h. After drug treatment, the absorbance of MTT dye was measured at 540 nm with a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).
For colony formation assays, the cells were seeded at a density of 3,000 cells per well in six-well plates and treated with targeted agents. After 12 days, the cell colonies were stained with 0.1% Coomassie Blue solution (Sigma-Aldrich, St. Louis, MO, USA), and counted using a Gel Doc (Bio-Rad, Hercules, CA, USA). The data presented are representative of three independent experiments.

Nam A.R., Kim J.W., Cha Y., Ha H., Park J.E., Bang J.H., Jin M.H., Lee K.H., Kim T.Y., Han S.W., Im S.A., Kim T.Y., Oh D.Y, & Bang Y.J. (2016). Therapeutic implication of HER2 in advanced biliary tract cancer. Oncotarget, 7(36), 58007-58021.

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Gelatine Zymography for MMP Activity

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Gelatine zymography was performed to determine matrix metalloproteinases (MMP) activity in cells under normoxia, submitted to hypoxia and exposed to Dfx. Proteins (15 μg/lane) from conditioned media were separated on 10% polyacrylamide zymogram gels with 0.1% gelatine (MERCK) as substrate. After electrophoresis, gels were incubated in 2% Triton X-100 (Sigma) in Milli-Q water for protein renaturation, with gentle agitation for 30 min at room temperature. Subsequently, gels were incubated in MMP substrate buffer (50 mM Tris-HCl, pH 7.5; 10 mM CaCl₂) overnight with gentle shaking at 37°C. The gels were finally stained with filtered Coomassie blue solution (Sigma) for 30 min and then washed with deionized water until the adequate resolution was obtained. Gelatinolytic bands were observed as white areas against the blue background, and the intensity of the bands was evaluated using the Quantity One Software (Biorad).

Peixoto A., Fernandes E., Gaiteiro C., Lima L., Azevedo R., Soares J., Cotton S., Parreira B., Neves M., Amaro T., Tavares A., Teixeira F., Palmeira C., Rangel M., Silva A.M., Reis C.A., Santos L.L., Oliveira M.J, & Ferreira J.A. (2016). Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension. Oncotarget, 7(39), 63138-63157.

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Comparative Drug Response Assay

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To compare the drug response between parental cells and resistant cells, the cells were seeded into six-well plates and incubated with the indicated concentration of each drug for 14 days. The cell colonies were washed in phosphate-buffered saline and stained with 0.1% Coomassie Blue solution (Sigma Aldrich) for 1 h at room temperature. The excess staining solution was then removed, and the plates were washed in PBS and air-dried. The cell colonies were counted using GELCOUNT (Oxford Optronix Ltd., Abingdon, UK), and cell viability was calculated by using SigmaPlot.

Kim J.W., Min A., Im S.A., Jang H., Kim Y.J., Kim H.J., Lee K.H., Kim T.Y., Lee K.W., Oh D.Y., Kim J.H, & Bang Y.J. (2020). TDP1 and TOP1 Modulation in Olaparib-Resistant Cancer Determines the Efficacy of Subsequent Chemotherapy. Cancers, 12(2), 334.

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Zymographic Analysis of MMP-1 Protease Activity

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Protease activity of MMP-1 was detected by zymographic analysis. Samples were homogenized in the RIPA buffer without PMSF. Equal amounts of protein (20 µg) were prepared in nondenaturing sample buffer and loaded into the well of the SDS-PAGE gel containing 1 mg/mL gelatin (Sigma-Aldrich, St. Louis, MO, USA). gelatin is not only degraded by gelatinases (MMP-2 and -9), but to a lesser extent by collagenase (MMP-1) [32 (link),50 (link),51 (link)]. After electrophoresis, gels were washed 2 × 15 min in 2.5% Triton X-100 solution to remove SDS. Then, gels were incubated with zymographic development buffer (50 mM Tris, pH 7.4, 10 mM CaCl₂, and 0.05% Brij 35) overnight at 37 °C and stained with Coomassie blue solution (Sigma-Aldrich, St. Louis, MO, USA). Cleared areas indicated proteolysis of the gelatin. Zymograms were quantified by densitometry with the Gel Doc 1000 system (Bio-Rad Laboratories, Hercules, CA, USA) and presented as the fold of the control value.

Zhang Z.K., Li J., Yan D.X., Leung W.N, & Zhang B.T. (2016). Icaritin Inhibits Collagen Degradation-Related Factors and Facilitates Collagen Accumulation in Atherosclerotic Lesions: A Potential Action for Plaque Stabilization. International Journal of Molecular Sciences, 17(2), 169.

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MTT and Colony Formation Assays for Jab1

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For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, cells transfected with Jab1-specific or control siRNA or shRNA were seeded in 96-well plates: 2,000 cells per well for the SNU478 cell line and 4,000 cells per well for the HuCCT-1 cell line. MTT dye (Sigma-Aldrich) was added to each well and cells were incubated for 4 hours at 37°C. The MTT solution was removed and dimethyl sulfoxide was carefully added. Cell viability was calculated by measuring the absorbance at 540 nm with a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA). Six wells were used for each experimental condition.
For colony formation assays, SNU478 cells and HuCCT-1 cells were seeded in six-well plates at a density of 500 cells and 3,000 cells, respectively. The cells were then treated with Jab1-specific or control siRNAs. After 10 days, the cell colonies were stained with 0.1% Coomassie Blue solution (Sigma-Aldrich), and counted using a Gel Doc (Bio-Rad, Hercules, CA). The data presented are representative of three independent experiments.

Nam A.R., Kim J.W., Park J.E., Bang J.H., Jin M.H., Oh D.Y, & Bang Y.J. (2018). Jab1 Silencing Inhibits Proliferation and Sensitizes to Cisplatin in Biliary Tract Cancer. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 51(3), 886-900.

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Colony Formation Assay of AZD1208

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For the colony formation assay (CFA), cells were seeded into 6-well plates and incubated for 48 hours at 37°C in 5% CO₂. Next, the cells were treated with various concentrations (0.1, 0.5, 1, 2.5, and 5 μmol/L) of AZD1208 alone or in combination with 100 nmol/L AZD5363 every 3 days. The cells were cultured until colonies formed (14 days). The colonies were stained with a 0.1% Coomassie blue solution (Sigma-Aldrich) and counted using a GelCount automatic plate scanner (Oxford Optronics GelCount, Oxford, UK). The cell survival rate and IC₅₀ of AZD1208 were determined using SigmaPlot software (SPSS Inc.).

Lee M., Lee K.H., Min A., Kim J., Kim S., Jang H., Lim J.M., Kim S.H., Ha D.H., Jeong W.J., Suh K.J., Yang Y.W., Kim T.Y., Oh D.Y., Bang Y.J, & Im S.A. (2018). Pan-Pim Kinase Inhibitor AZD1208 Suppresses Tumor Growth and Synergistically Interacts with Akt Inhibition in Gastric Cancer Cells. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 51(2), 451-463.

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Gelatin-SDS PAGE for MMP Activity

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Conditioned medium was replaced by a serum-free medium 24 h prior to experiment endpoint. The media were loaded into gelatin–SDS polyacrylamide gels (Mini-Protean Tetra Cell system, Bio-Rad), as described previously45 (link). Following electrophoresis, gels were stained with 0.1% w/v Coomassie Blue solution (Sigma-Aldrich). Clear bands of proteolytic degradation in contrast to a blue background of the gelatin substrate represent the activity of MMPs. Band densities were quantified by densitometric analysis using ImageJ software.

Sousa D.M., Conceição F., Silva D.I., Leitão L., Neto E., Alves C.J., Alencastre I.S., Herzog H., Aguiar P, & Lamghari M. (2016). Ablation of Y1 receptor impairs osteoclast bone-resorbing activity. Scientific Reports, 6, 33470.

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Clonogenic Potential of CD44+ Cells

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To determine the clonogenic potential of CD44⁺ cells, a colony-forming assay was performed, as previously described [32 (link)]. Briefly, CD44⁺ cells were seeded in a 32mm Petri dish at a density of 1000 cells/plate. After 7 days, the colonies were stained with 0.1% of a Coomassie Blue solution (Sigma−Aldrich).

Jaksic Karisik M., Lazarevic M., Mitic D., Nikolic N., Milosevic Markovic M., Jelovac D, & Milasin J. (2023). Osteogenic and Adipogenic Differentiation Potential of Oral Cancer Stem Cells May Offer New Treatment Modalities. International Journal of Molecular Sciences, 24(5), 4704.

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Quantification of Secreted MMPs in Cell Culture

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Cell culture supernatants were analyzed for the presence of secreted MMPs by gelatine-zymography on day 14. Before sampling, cell-laden hydrogels were maintained overnight in serum-free medium. The conditioned media were collected and centrifuged (10000 rpm, 5 min) to remove cell debris, and loaded into gelatin-SDS polyacrylamide gels. Sample volumes were adjusted to yield equivalent total protein contents in the supernatant, which were quantified using the DC Protein assay (Bio-Rad). A protein ladder was run as a reference. The gel was run in 1x Tris–Glycine SDS running buffer at 80 V (Mini-Protean Tetra Cell system, Bio-Rad). After electrophoresis, gels were washed twice with 2% v/v Triton X-100 and incubated in MMP substrate buffer (50 mM Tris–HCl, pH 7.5, 10 mM CaCl2) for 16 h at 37 °C. Thereafter, gels were washed and stained with 0.1% w/v Coomassie Blue solution (Sigma). MMP proteolytic activity was visualized as clear bands against a blue background of stained gelatin substrate. MMP quantification was performed using Fiji software [21 (link)].

Neves M.I., Bidarra S.J., Magalhães M.V., Torres A.L., Moroni L, & Barrias C.C. (2023). Microstructured click hydrogels for cell contact guidance in 3D. Materials Today Bio, 19, 100604.

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Synthesis and Characterization of Gold Nanoparticles

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Hydrogen tetrachloroaurate(III) trihydrate (HAuCl 4 •4H 2 O) and Costar Clear Polystyrene 96-Well Plates were purchased from Fisher Chemical. Ascorbic acid, AgNO 3 , cetyltrimethylammonium bromide (CTAB), 10 nm gold colloid suspensions (6×10 12 /mL), HEPES buffer and Coomassie Blue solution were purchased from Sigma Aldrich. Ultrapure deionized water (resistivity greater than 18.0 M.Ω.cm -1 ) was used for all solution preparations and experiments.

Sojinrin T., Conde J., Liu K., Curtin J., Byrne H.J., Cui D, & Tian F. (2017). Plasmonic gold nanoparticles for detection of fungi and human cutaneous fungal infections. Analytical and bioanalytical chemistry, 409(19).

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