The koji extract was the same sample as in the glucose uptake assay. Monacolin K, manscin, or citrate-Na buffer (pH 7.4) were also prepared to exposure for cells; monacolin K solutions (1–1000 µM), monascin solution (5–20 µM), and citrate-Na buffer (pH 7.4) (3–150 mM), respectively. Cells (1 × 105 cells) were grown in 10-cm petri dishes with 5 ml DMEM/high glucose medium and 0.1 ml samples for 14 h. The L6 myotube cells were washed 3 times in PBS, trypsinized, and the trypsin was removed by centrifugation. Protein extracts were prepared by homogenizing the tissue in 50 mM Tris–HCl (pH 7.6) and 0.1% Triton X-100 supplemented with a protease inhibitor cocktail (F. Hoffmann-La Roche Ltd., Mannheim, Germany) and centrifuged at 21,500 × g for 30 min. The supernatant was used as the crude extract. The protein concentrations of extracts were determined using the Coomassie Protein Assay Reagent Kit (Thermo Fisher Scientific, Inc., CA, USA). Samples containing 60 µg of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels and transferred to polyvinylidene fluoride (PVDF) membranes (Hybond-P; GE Healthcare UK Ltd., Buckinghamshire, UK). Membranes were probed with glucose transporter type 4 (GLUT4) and β-actin antibodies. The signal intensities were quantitated using the NIH Image J software (http://rsb.info.nih.gov/nih-image ).
Coomassie protein assay reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Coomassie protein assay reagent kit is a colorimetric assay used to quantify the total protein concentration in a sample. The kit contains reagents that bind to proteins, resulting in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Empty gravity ow column, by Bio-Rad (3 mentions)
Anti dykddddk g1 a nity resin, by GenScript (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ecl prime western blot detection kit, by GE Healthcare (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Hybond p, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Hybond, by Cytiva (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Reduced glutathione gsh colorimetric assay kit, by Elabscience (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
16 protocols using coomassie protein assay reagent kit
1
Glucose Uptake Assay with Koji Extract
2
Testicular Protein Extraction and Western Blotting
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Testicular tissues or purified germ cells were homogenized at 4°C in 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, supplemented with 1 µg/ml
leupeptin, 1 µg/ml pepstatin A, 1 mM DTT, and 0.5 mM phenylmethanesulfonyl fluoride (PMSF), using a Teflon-glass homogenizer (750 rpm, 10 strokes). After
incubation at 4°C for 4 h, the homogenates were centrifuged at 13,400 × g for 10 min at 4°C. The supernatant solutions were used as protein
extracts. Protein concentration was determined by using a Coomassie protein assay reagent kit (Thermo Fisher Scientific). Protein samples (5 µg) were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore). After blocking with 2%
skim milk or gelatin, the blots were probed with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA). The immunoreactive bands were visualized by an ECL or an ECL Prime Western blot detection kit (GE Healthcare).
leupeptin, 1 µg/ml pepstatin A, 1 mM DTT, and 0.5 mM phenylmethanesulfonyl fluoride (PMSF), using a Teflon-glass homogenizer (750 rpm, 10 strokes). After
incubation at 4°C for 4 h, the homogenates were centrifuged at 13,400 × g for 10 min at 4°C. The supernatant solutions were used as protein
extracts. Protein concentration was determined by using a Coomassie protein assay reagent kit (Thermo Fisher Scientific). Protein samples (5 µg) were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore). After blocking with 2%
skim milk or gelatin, the blots were probed with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA). The immunoreactive bands were visualized by an ECL or an ECL Prime Western blot detection kit (GE Healthcare).
3
Recombinant Protein Purification from E. coli
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The pGEX-6P-2 plasmids with LDH2 cDNA were transformed into E. coli DH5α-competent cells and grown in LB medium supplemented with 50 μg/ml ampicillin with vigorous shaking at 37°C up to an optical density of 0.6 measured at 600 nm. Then, the GST-fused protein was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM with mild shaking at 25°C overnight. The cells were centrifuged and the bacterial pellet was resuspended in phosphate buffered saline (PBS) containing 10μg/ml of lysozyme, and then stored at -20°C. After thawing, the cells were disrupted by sonication on ice, cell debris was removed by centrifugation (3,500 rpm, 20 min), and the resulting supernatant was recovered to a new tube. Recombinant proteins in the soluble fraction were affinity purified by glutathione-Sepharose beads according to the manufacturer’s protocols (GE Healthcare, Buckinghamshire, UK). Beads were washed three times with PBS and then bound proteins were eluted with elution buffer (200 mM NaCl, 20 mM reduced glutathione, 100 mM Tris-HCl [pH 9.5] and 5 mM EDTA). The eluted fractions were dialyzed against PBS and the amount of recombinant proteins were calculated using the Coomassie protein assay reagent kit according to the manufacturer’s protocols (Thermo Scientific, MA, USA).
4
Induction and Enzymatic Assay of Bacterial pdef
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Bacterial strains containing the pdef plasmid (AS192 or AS194) or mutant pdef derivatives (AS348 or AS349) were grown overnight in 5 ml LB medium and then diluted 1:100 in 50 ml fresh LB medium. Cells were induced for 1 h with 0.5 mM IPTG at an OD600 of 0.5 to 0.6. Cells were harvested, washed once with assay buffer (50 mM HEPES [pH 7.4] and 25 mM NaCl), and resuspended in 0.5 ml assay buffer containing 0.1 mM diethylenetriamine penta-acetic acid (DTPA). Cells were lysed mechanically using 0.1-mm silica beads, and cell debris was removed by centrifugation. The clear cell lysate was immediately used for enzyme assays. The protein content of cell lysates was estimated using the Coomassie protein assay reagent kit (Thermo Fisher Scientific). To measure the effects of zinc or NO· on enzyme activity, 125 μM ZnSO4 or 25 to 75 μM spermine NO· were added during the IPTG induction phase.
5
Tissue and Cell Homogenization Protocol
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Male C57BL/6 mouse tissues (4-month-old) and transfected HEK293T cells were homogenized at 4°C in buffer A containing 20 mM Tris/HCl, pH 7.5, 0.15 M NaCl, and
0.5% Nonidet P-40, supplemented with 0.5 mM dithiothreitol (DTT), 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 0.5 mM phenylmethanesulfonyl fluoride (PMSF), in a
Teflon-glass homogenizer (750 rpm, 10 strokes). The homogenates were centrifuged at 13,400 × g for 10 min at 4°C to remove cellular debris.
Protein concentration of the supernatants was determined using a Coomassie protein assay reagent kit (Thermo Fisher Scientific).
0.5% Nonidet P-40, supplemented with 0.5 mM dithiothreitol (DTT), 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 0.5 mM phenylmethanesulfonyl fluoride (PMSF), in a
Teflon-glass homogenizer (750 rpm, 10 strokes). The homogenates were centrifuged at 13,400 × g for 10 min at 4°C to remove cellular debris.
Protein concentration of the supernatants was determined using a Coomassie protein assay reagent kit (Thermo Fisher Scientific).
6
Anti-inflammatory Molecular Mechanisms
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Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), glutaMAX, and penicillin-streptomycin (PS) were obtained from Gibco (Grand Island, NY, USA). Coomassie protein assay reagent kit and Minute™ Cytoplasmic and Nuclear Extraction Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The COX-1, COX-2, and NF-κB p65 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The iNOS and TNF-α antibodies were provided by Proteintech (Rosemont, IL, USA). AmershamTM ECL SelectTM Western Blotting Detection Reagent and AmershamTM Hybond P Western blotting membranes (PVDF) were purchased from Merck (Darmstadt, Germany). The β-Actin, RIPA lysis buffer, and secondary antibodies were obtained from Merck Millipore (Burlington, MA, USA). Prostaglandin E2 (PGE2) Express ELISA Kit (500141) was provided by Cayman Chemical (Ann Arbor, MI, USA). Reduced Glutathione (GSH) Colorimetric Assay Kit was obtained from Elabscience Biotechnology (Houston, TX, USA). Protease inhibitor cocktail was proved by Fivephoton Biochemicals (San Diego, CA, USA). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), indomethacin (IND), sodiumdodecyl sulfate (SDS), trypsin-EDTA, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Testicular Protein Extraction and Western Blot
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Testicular tissues were homogenized at 4°C in 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 0.5% Nonidet
P-40, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 0.5 mM phenylmethanesulfonyl fluoride, using a Teflon-glass
homogenizer (750 rpm, 10 strokes). After incubation at 4°C for 4 h, the homogenates were centrifuged in a
microcentrifuge at 13,400 × g for 10 min at 4°C. The supernatant solution was used as protein
extracts. Protein concentration was determined by means of the Coomassie Protein Assay Reagent Kit (Thermo
Fisher Scientific). Protein samples (5 μg) were subjected to SDS-polyacrylamide gel electrophoresis and
transferred onto polyvinylidene difluoride membranes (Merck Millipore). After blocking with 2% skim milk or
gelatin, the blots were probed with primary antibodies followed by incubation with horseradish
peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The
immunoreactive proteins were visualized by an ECL or an ECL Prime Western Blot Detection Kit (GE
Healthcare).
P-40, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 0.5 mM phenylmethanesulfonyl fluoride, using a Teflon-glass
homogenizer (750 rpm, 10 strokes). After incubation at 4°C for 4 h, the homogenates were centrifuged in a
microcentrifuge at 13,400 × g for 10 min at 4°C. The supernatant solution was used as protein
extracts. Protein concentration was determined by means of the Coomassie Protein Assay Reagent Kit (Thermo
Fisher Scientific). Protein samples (5 μg) were subjected to SDS-polyacrylamide gel electrophoresis and
transferred onto polyvinylidene difluoride membranes (Merck Millipore). After blocking with 2% skim milk or
gelatin, the blots were probed with primary antibodies followed by incubation with horseradish
peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The
immunoreactive proteins were visualized by an ECL or an ECL Prime Western Blot Detection Kit (GE
Healthcare).
8
Anticancer Compound Evaluation Protocol
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Roswell Park Memorial Institute (RPMI) Medium 1640, fetal bovine serum (FBS), and penicillin-streptomycin (PS) were purchased from Gibco (Grand Island, NY, USA). Bovine serum albumin (BSA) and a Coomassie protein assay reagent kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The Bax, Bcl-xL, Cyclin B1, and MMP-2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The MMP-9 antibody was provided by Genetex (Irvine, CA, USA). The β-actin, Cdc2, and Cdc25c were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The QIAamp DNA Mini Kit was obtained from Qiagen (Hilden, Germany). The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Propidium iodode (PI), sodiumdodecyl sulfate (SDS), and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). TAN, 5-DTAN, and 5-ATAN were provided by Dr. Chi-Tang Ho (Department of Food Science, Rutgers University, New Brunswick, NJ, USA).
9
Quantification of FucA Enzyme Activity
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Samples from fermentation broth were withdrawn, adjusted to a final OD600 of 4, centrifuged, and subsequently processed as described elsewhere (Durany et al., 2005 (link); Vidal et al., 2008 (link)). Briefly, pellets were resuspended in 100 mM Tris HCl (pH 7.5). Cell suspensions were placed in ice and sonicated [VibracellTM model VC50 (Sonics & Materials)], over four pulses of 15 s each at 50 W with 2 min intervals in ice between each pulse. Cellular debris was then removed by centrifugation, and the cleared supernatant was collected for FucA analysis. One unit of FucA activity is defined as the amount of enzyme required to convert 1 μmol of fuculose-1-phosphate in DHAP and L-lactaldehyde for minute at 25 °C and pH 7.5 (Vidal et al., 2008 (link)).
Total protein content was determined by means of the Bradford method using a Coomassie® Protein Assay Reagent Kit (Thermo Scientific). To quantify the amount of FucA relative to total intracellular soluble proteins, NuPAGE® 12% Bis–Tris gels were used according to the manufacturer's instructions (Invitrogen). Protein concentration was quantified using Kodak Digital Science® 1D 3.0.2 densitometry software.
Average values of triplicates experiments were plotted with error bars. The error indicates the confidence interval of 90%.
Total protein content was determined by means of the Bradford method using a Coomassie® Protein Assay Reagent Kit (Thermo Scientific). To quantify the amount of FucA relative to total intracellular soluble proteins, NuPAGE® 12% Bis–Tris gels were used according to the manufacturer's instructions (Invitrogen). Protein concentration was quantified using Kodak Digital Science® 1D 3.0.2 densitometry software.
Average values of triplicates experiments were plotted with error bars. The error indicates the confidence interval of 90%.
10
Extracting Fish Myofibrillar Proteins
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For fish myofibrillar protein extraction, about 50 mg sample was homogenized with 1 mL buffer (5% NaCl, 0.02 M Tris-HCl, pH 7.2) and 0.1% protease inhibitor cocktail (Nacalai Tesque, Inc., Japan). The supernatant was obtained by centrifugation at 0°C, 13,416×g for 20 mins. The protein concentration was determined by the Coomassie protein assay reagent kit (Thermo Fisher Scientific, USA) following the manufacturer's instructions.
A total of 5 µg of protein were treated with the sample buffer solution with and without 2x 2-Mercaptoethanol (2-ME; Nacalai Tesque, Inc., Japan) for SDS-PAGE and electrophoresed in 10% polyacrylamide gel by Tricine SDS-PAGE system (Schägger and von Jagow, 1987) (link). The protein bands were stained with 0.04% Coomassie Brilliant Blue R-250 in 40% methanol and 10% acetic acid and destained with 10% acetic acid.
A total of 5 µg of protein were treated with the sample buffer solution with and without 2x 2-Mercaptoethanol (2-ME; Nacalai Tesque, Inc., Japan) for SDS-PAGE and electrophoresed in 10% polyacrylamide gel by Tricine SDS-PAGE system (Schägger and von Jagow, 1987) (link). The protein bands were stained with 0.04% Coomassie Brilliant Blue R-250 in 40% methanol and 10% acetic acid and destained with 10% acetic acid.
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