Vastus lateralis muscles were sectioned with a Leica CM 1900 cryostat (Walldorf, Baden-Wurttemberg, Germany). Thirty 10 μm cryosections were lysed in 50 μL of RIPA buffer (0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, 150 mM sodium chloride, and 50 mM TrisHCl pH 7.5) for 30 minutes on ice, with protease inhibitor cocktail (Complete, Roche, Mannheim, Germany) as well as phosphatase inhibitor cocktail (PhosStop, Roche, Mannheim, Germany). At the end of the incubation, the cell extracts were centrifuged for 10 minutes (12,000 g) at 4°C. The amount of protein was calculated using the Quick Start Bradford Protein Assay Kit 1 (Bio Rad Laboratories, Hercules, CA). Then 30 μg of protein in NuPAGE LDS Sample Buffer (Life Technologies, Grand Island, NY) and NuPAGE Sample Reducing Agent (Life Technologies, Grand Island, NY) was loaded to SDS-PAGE gel for immunoblotting analysis. The primary antibodies used were pSTAT3 (Y705, 1 : 1000; Cell Signaling Technology, Danvers, MA), pSTAT3 (S727, 1 : 1000; Cell Signaling Technology, Danvers, MA), and Total STAT3 (1 : 1000; Cell Signaling Technology, Danvers, MA). Bound antibodies were detected using ECL reagents. The results were normalized to GAPDH (1 : 5000; Millipore, Billerica, MA). Band intensity was evaluated by densitometry analysis, normalized to its total content, and reported as fold increase relative to respective control set as 1.
Quick start bradford protein assay kit 1
Manufactured by Bio-Rad
Sourced in United States
The Quick Start Bradford Protein Assay Kit is a colorimetric assay used to determine the concentration of protein in solution. It utilizes the Bradford method, which involves the binding of Coomassie Brilliant Blue G-250 dye to proteins, resulting in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Total stat3, by Cell Signaling Technology (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Gapdh, by Merck Group (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Cm1900, by Leica (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Multiscan sky, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
2 d clean up kit, by GE Healthcare (1 mentions)
Protease inhibitor mini tablet, by Thermo Fisher Scientific (1 mentions)
550 sonic dismembrator, by Thermo Fisher Scientific (1 mentions)
6 protocols using quick start bradford protein assay kit 1
1
Immunoblotting Analysis of pSTAT3 Signaling
2
Quantification of Exosomal Protein Content
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Exosomes were quantified on protein content bases using the Bradford standard protein assay. We diluted 2.0 µL of the samples in 38 µL of deionized distilled water to obtain a final volume of 40 µL in a 96-well plate. Then, 10 µL of the dilute samples were placed in each well of 96-well plates, and 190 mL of 1 × Bradford assay reagent was added (Quickstart, Bradford Protein Assay Kit 1, BioRad, Hercules, CA, USA) and incubated for 5 min. Next, the OD value was recorded using a microplate reader (Multiscan Sky, Thermoscientific) at 595 nm wavelength. Bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) 50 µg/µL was run as the standard control.
3
Protein Concentration Determination Using Bradford Assay
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The protein concentrations were determined as described by Bradford (1976) (link), using a Quick Start Bradford Protein Assay Kit 1 (500–0201; Bio-Rad, United States Laboratories) and bovine serum albumin (BSA) as a standard (Bio-Rad Laboratories, United States).
4
Quantitative Proteome Analysis of DENV2-Infected HepG2 Cells
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HepG2 cells were seeded overnight at a density of 1 × 106 cells per 25 cm2 culture flasks and then inoculated with DENV2 at the optimized MOI of 15. The viral inoculum was removed after 2 h of infection and the cells were further incubated at the optimized time-point (24 h) in DMEM maintenance medium containing the optimized concentration of 5 µg/ml YK51 compound. Samples with different conditions were also cultured accordingly and processed in the same manner. Subsequently, the cells were harvested for protein extraction. The whole cell proteome was extracted in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% IPG buffer, 40 mM DTT) on ice for 30 min. The extracted proteins were centrifuged at 13,000 rpm for 5 min to remove cellular debris. The resulting supernatant was cleaned using 2-D clean-up kit (GE Heathcare, Danderyd, Sweden) following the manufacturer’s instructions. Quick Start™ Bradford protein assay kit 1 (Bio-Rad Laboratories, Hercules, CA, USA) was then used to quantify the protein concentration.
5
Quantifying Fungal Protein Production
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A synthetic agar medium DFM—with double glucose content as the only added carbon source and no added nitrogen, supplemented with gentamicin at 0.512 mg/L—was used to keep the S. maltophilia from growing on the medium alone. The plates were incubated at 25 for 15–20 days with a 12 h light and dark photoperiod. The Bradford protein assay was used to determine the protein amount of Fg-S. maltophilia and PH-1 cultures on the medium without added a nitrogen source. Protein was extracted from a standardised area of the mycelium using a cork plug, and total proteins were extracted. The amount of Coomassie 1 Brilliant Blue G-250 dye colouring the extracted proteins (Bradford 1976) was then used to measure total protein content. A dilution series of bovine serum albumin (BSA) 2 mg/mL solution was used as a standard reference. The procedure followed the Quick Start Bradford Protein Assay Kit 1 (Bio-Rad, Beijing, China) protocol.
6
Protein Extraction and Purification
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Cell pellets from 20 mL of culture were resuspended in 3 mL of buffer containing 2% Triton-X-100, 65 mM DTT, 10 mM EDTA, 20 mM Tris-HCl pH 8.0, and 15 mg of Protease Inhibitor Mini Tablets, EDTA-free (Thermo Fisher Scientific, Waltham, MA, USA). Cells were disrupted on the Sonic; Dismembrator 550 (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at 35% intensity. Cell debris and ribosomal fraction were precipitated by ultracentrifugation at 213,000× g, 4 °C for 30 min. Proteins were precipitated with the addition of 1:1 (V:V) of 20% TCA and 0.2% DTT in ice-cold acetone to supernatant, and the resulting mixture was then incubated at 4 °C overnight. Denatured proteins were centrifuged at 20,000× g, 4 °C for 15 min. The pellet was washed 3 times with 0.2% DTT in ice-cold acetone followed by 1 wash with Milli-Q water, air dried, and dissolved in 40 µL of buffer containing 7 M urea, 2 M thiourea, 4% (W:V) CHAPS, 65 mM DTT, 20 mM Tris-HCl, pH 8.5. Protein concentration was measured using the Quick Start™ Bradford Protein Assay Kit 1 (Bio-Rad, Berkeley, CA, USA) and validated using SDS-PAGE.
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