Protein was extracted from isolated abdominal aortas that had been snap frozen in liquid nitrogen and homogenized in a buffer containing 1 M NaCl, 2 M urea, 0.2 mM PMSF, 50 mM Tris (pH 7.4), 0.1% EDTA, 0.1% Brij-35, and protease inhibitors (10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin), as previously described41 (link). Samples were sonicated on ice and centrifuged, and supernatants were used to quantify protein content. Protein lysate was placed in a nonreducing zymogram buffer (Cat#161–0764, Bio-Rad, Hercules, CA) and applied without boiling to a 10% zymogram gel (#161–1167, Bio-Rad). Gels were incubated in 2% Triton X-100 at room temperature for 30 min and then rinsed in H2O for 5 min. Gels were incubated overnight at 37°C with gentle agitation in Zymogram developing buffer (Cat# 161–0766, Bio-Rad) containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35. Proteins were stained with Coomassie brilliant blue R-250 solution (Cat# 161–0436, Bio-Rad) and destained with a solution containing 40% methanol, 10% acetic acid, and 50% H2O.
Coomassie brilliant blue r 250 solution
Manufactured by Bio-Rad
Sourced in United States
Coomassie Brilliant Blue R-250 solution is a laboratory reagent used for the detection and quantification of proteins in various applications, such as gel electrophoresis and protein assays. It is a dye-based staining solution that binds to proteins, resulting in the formation of a blue-colored complex that can be detected and measured.
Automatically generated - may contain errors
Lab products found in correlation
Zymogram gel, by Bio-Rad (3 mentions)
Zymogram developing buffer, by Bio-Rad (1 mentions)
Perfection v850 pro photo scanner, by Epson (1 mentions)
Labscan, by RCI Labscan (1 mentions)
Versadoc imager, by Bio-Rad (1 mentions)
Prestained molecular weight markers, by Bio-Rad (1 mentions)
Thiamine hcl, by Merck Group (1 mentions)
Bis acrylamide, by Bio-Rad (1 mentions)
Acrylamide, by Bio-Rad (1 mentions)
Ammonium persulfate, by Bio-Rad (1 mentions)
Myo inositol, by Merck Group (1 mentions)
Restriction endonucleases, by Roche (1 mentions)
Temed, by Bio-Rad (1 mentions)
Gateway bp, by Thermo Fisher Scientific (1 mentions)
Mini protean 3 cell, by Bio-Rad (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Cefotaxime, by Duchefa Biochemie (1 mentions)
Phosphinothricin, by Duchefa Biochemie (1 mentions)
T4 dna ligase, by Roche (1 mentions)
Tetracycline, by Duchefa Biochemie (1 mentions)
16 protocols using coomassie brilliant blue r 250 solution
1
Protein Extraction and Zymogram Analysis
2
Proteome Labeling with Acrylate Alkyne Warhead
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The performance of compound 14 was compared to that
of the reported acrylate alkyne warhead oct-1-en-7-yn-3-one.58 (link) The prepared mouse proteome (2 μL, 23.7
μg/μL) was diluted with water (10 μL), and the mixture
incubated with the warhead (1 μL, 200 μM) at room temperature
for 2 h. After this time, the solution was incubated with rhodamine
110-azide (1 μL, 200 μM), copper(II) sulfate (1 μL,
25 mM), and sodium ascorbate (1 μL, 25 mM) for 2 h. Each solution
was further incubated with a nonreducing, fluorescent compatible sample
buffer (5 μL, Fisher Scientific). The mixture was heated at
98 °C for 5 min and then allowed to cool to room temperature.
As a reference for the bands, the Precision Plus Protein Unstained
Protein Standard Ladder (Bio-Rad) was used. The prepared samples were
then separated by 4% to 20% mini-PROTEAN TGX stain free SDS–PAGE
(Bio-Rad) analysis. The gel was rinsed with water, and the bands were
visualized with an Amersham Imager 680 instrument (λex = 492 nm; λem = 508 nm). To verify the presence
of the proteome, the gel was stained with a Coomassie Brilliant Blue
R-250 solution (Bio-Rad). The gel was washed three times with water,
and the bands were visualized with an Amersham Imager 680 instrument.
Final concentrations within this assay are 2.37 μg/μL
proteome, 10 μM warhead, 10 μM rhodamine dye, 625 μM
copper(II) sulfate, and 625 μM sodium ascorbate.
of the reported acrylate alkyne warhead oct-1-en-7-yn-3-one.58 (link) The prepared mouse proteome (2 μL, 23.7
μg/μL) was diluted with water (10 μL), and the mixture
incubated with the warhead (1 μL, 200 μM) at room temperature
for 2 h. After this time, the solution was incubated with rhodamine
110-azide (1 μL, 200 μM), copper(II) sulfate (1 μL,
25 mM), and sodium ascorbate (1 μL, 25 mM) for 2 h. Each solution
was further incubated with a nonreducing, fluorescent compatible sample
buffer (5 μL, Fisher Scientific). The mixture was heated at
98 °C for 5 min and then allowed to cool to room temperature.
As a reference for the bands, the Precision Plus Protein Unstained
Protein Standard Ladder (Bio-Rad) was used. The prepared samples were
then separated by 4% to 20% mini-PROTEAN TGX stain free SDS–PAGE
(Bio-Rad) analysis. The gel was rinsed with water, and the bands were
visualized with an Amersham Imager 680 instrument (λex = 492 nm; λem = 508 nm). To verify the presence
of the proteome, the gel was stained with a Coomassie Brilliant Blue
R-250 solution (Bio-Rad). The gel was washed three times with water,
and the bands were visualized with an Amersham Imager 680 instrument.
Final concentrations within this assay are 2.37 μg/μL
proteome, 10 μM warhead, 10 μM rhodamine dye, 625 μM
copper(II) sulfate, and 625 μM sodium ascorbate.
3
Protein Visualization by Coomassie Staining
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Protein samples (1 µg each) were loaded on the SDS-PAGE. After the electrophoresis run, the gel was stained with Coomassie Brilliant Blue R-250 solution (1610436, Bio-Rad, Hercules, CA, USA) for 30 min with gentle agitation followed by washing with the destaining solution (50% methanol, 10% glacial acetic acid) for 2 h until the background became less dark.
4
Bambara Bean Protein SDS-PAGE Analysis
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the procedures of Laemmli (1970) (link). The gel system consisted of 12% (w/v) polyacrylamide for the resolving gel (pH 8.8) and 4.5% (w/v) for the stacking gel (pH 6.8). Five milligrams of both flour and the protein isolate of Bambara beans were suspended in 1 mL of distilled water and diluted 25 times before loading. Samples were prepared under reducing conditions using β-mercapto ethanol, heated at 95 °C in a water bath for 5 min, and then centrifuged for 5 min at 5000 rpm. Fifteen microliters of each sample were loaded into the wells. Electrophoretic separation was carried out at 70 V for the stacking gel and at 100 V for the resolving gel. Protein bands were fixed by immersion of the gel in a 10% (v/v) acetic acid solution for 30 min, stained in a 0.1% (w/v) Coomassie brilliant blue R-250 solution (Bio-Rad) for 30 min and then distained in 10% (v/v) acetic acid and 10% (v/v) methanol for 40 min.
5
Coomassie Staining of Cultured Cells
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LAUD cells were plated in 6-well culture plates (5 × 103 cells/well). The medium was changed every 3 days. After 10 days, the cells were stained with a 0.005% Coomassie Brilliant Blue R-250 solution (Bio-Rad, USA), and the plates were imaged using an Epson Perfection V850 Pro Photo Scanner (USA).
6
Fibroin Protein Separation by SDS-PAGE
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Protein in the isolated fibroin was separated using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method on a 5% stacking gel and a 12% separating gel. After electrophoresis, the gel was stained in Coomassie Brilliant Blue R-250 solution (BIO-RAD Laboratories) for 2 h and de-stained with a mixture of 7% acetic acid (VWR International Ltd., Poole, UK) and 10% methanol (RCI Labscan) in water. The molecular weights of protein bands were estimated by comparing them with standard molecular weight markers.
7
Nanoworm Purity and Transition Characterization
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Purity of the constructs was determined using SDS-PAGE. Briefly, 10–15 μg of protein was added to SDS page loading buffer and boiled at 95 °C for 5 min. The sample was electrophoresed on 4–20% precast SDS-PAGE gel. After the samples are run the gel was stained using 50 mL of Coomassie Brilliant Blue R-250 solution (Biorad). The gel was imaged on a Biorad Versadoc imager (Hercules, CA) using white light. The purity of samples was calculated using ImageJ. Briefly, pictures were imported into ImageJ and converted to 8-bit files. Individual lanes were selected, and an intensity profile was extracted. The peak areas were calculated, and the purity was determined using the following equation: where Apeak is the area of peak, and Atot is the area under all of the peaks.
The transition temperature was characterized to confirm that the nanoworms remain soluble under physiological temperatures. Transition temperature was determined using optical density measurements at 350 nm in PBS. Briefly, increasing concentrations of constructs were added to 300 μL Beckman coulter Tm microcells (Brea, CA), and the temperature was ramped at a rate of 1 °C/min. The optical density was plotted as a function of temperature, and the maximum first derivative of this curve was defined as the transition temperature.
The transition temperature was characterized to confirm that the nanoworms remain soluble under physiological temperatures. Transition temperature was determined using optical density measurements at 350 nm in PBS. Briefly, increasing concentrations of constructs were added to 300 μL Beckman coulter Tm microcells (Brea, CA), and the temperature was ramped at a rate of 1 °C/min. The optical density was plotted as a function of temperature, and the maximum first derivative of this curve was defined as the transition temperature.
8
Cetuximab and AREG Colony Assay
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Cells were seeded in 6-well plates at 1000 cells/well (30006, SPL Life Science, Pocheon, Korea). The treatments, 10 and 20 μg/mL of cetuximab and 50 ng/mL of AREG, were added three times weekly for 3 or 4 weeks to SNU-C4 or Caco-2, respectively. The cell colonies were stained with Coomassie brilliant blue R-250 solution (1610436, BioRad) for 2 h at room temperature. The representative colony images were photographed by ChemiDoc Touch Imaging System. The procedure of colony de-staining was performed using a 1% SDS solution to analyze the relative colony area. The relative colony area was determined at 595 nm using a Synergy H1 microplate reader. Protein expression was analyzed by ImageJ software (NIH, Baltimore, MD, USA).
9
Zymography for MMP-2 and MMP-9 Activity
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Matrix metalloproteinase (MMP)-2 and MMP-9 activity was measured by zymography as previously described [16 (link)]. In brief, protein lysate (600 µg) was placed in a non-reducing zymogram buffer and applied without boiling to a 10% zymogram gel (Bio-Rad). Gels were incubated in 2% Triton X-100 at room temperature for 30 min, and then rinsed in distilled water for 5 min. Gels were incubated overnight at 37°C with gentle agitation and proteins were stained with Coomassie Brilliant Blue R-250 solution (Bio-Rad) and de-stained with a solution containing 40% methanol, 10% acetic acid and 50% water.
10
Recombinant LTB and CTB Protein Production
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Murashige and Skoog (MS) medium, spectinomycin, and sucrose were purchased from MB cell (CA, U.S.A). Tetracycline, cefotaxime and phosphinothricin were purchased from Duchefa (Haarlem, Netherlands). The pMJ103 vector was constructed by GreenGene BioTech (Yongin, Korea) and the plasmids (MYO51, MYO53) containing LTB and/or CTB were kindly donated from Dr. M.S. Yang (Chonbuk National University). T4 DNA ligase and restriction endonucleases were obtained from Roche (Basel, Switzerland). Gateway BP and LR recombinase systems were purchased from Invitrogen (Breda, Netherlands). A mini trans-blot cell and Mini-Protean III cell were purchased from Bio-Rad (Hercules, CA, USA). Reagents for SDS-polyacrylamide electrophoresis, such as acrylamide, bis-acrylamide, ammonium persulfate, TEMED, prestained molecular weight markers and Coomassie Brilliant Blue R-250 solution were obtained from Bio-Rad (Hercules, CA, USA). Anti-LTB polyclonal antibody was obtained from Abcam (Cambridge, MA, USA). GM1-ganglioside and anti-rabbit IgG-conjugated alkaline phosphatase were purchased from Santa-Cruz Biotechnology (CA, USA). Thiamine-HCl, myo-inositol, anti-CTB polyclonal antibody, and other reagents such as salts and buffer components were of analytical grade and purchased from Sigma (St. Louis, MO, USA).
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