Cells were treated with LL-37 (0, 8, 16 µM) and lysed in radioimmunoprecipitation buffer containing 50 mM Tris, 150 mM NaCl, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM ethylenediaminetetraacetic acid, 0.1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride and 10 μg/ml aprotinin. The Coomassie Plus Protein Assay Reagent kit (#23238, Thermo Fisher Scientific) was used to quantify the proteins in the cell lysates. The total cellular proteins in the lysates were then separated by 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After conjugation with primary and secondary antibodies, immunoreactive bands were visualized using a chemiluminescence substrate. The intensity of each band was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States) and normalized to ß-actin housekeeping control.
Coomassie plus protein assay reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Coomassie Plus Protein Assay Reagent kit is a colorimetric assay used to quantify the total protein concentration in a sample. The kit contains a reagent solution that changes color in proportion to the amount of protein present in the sample. This allows for the determination of the total protein content through spectrophotometric analysis.
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Lab products found in correlation
3 protocols using coomassie plus protein assay reagent kit
1
Quantification of LL-37-Induced Protein Changes
2
Preparing Crude Worm Antigen and Excretory-Secretory Product
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Crude worm antigen (CWA) was prepared by homogenizing adult worms in phosphate buffered saline (PBS) containing 1% Triton X-100. After homogenization, the homogenate was sonicated on ice at an amplitude of 30% with a 9-s on/off pulse using an ultrasonic processor (Vibra-cell, Sonics, Newtown, CT, USA). The CWA was centrifuged at 12,000×g at 4 °C for 30 min and the clear supernatant was transferred into several microfuge tubes, followed by storage at − 80 °C until use. Excretory-secretory (ES) product was prepared from cultured adult worms in RPMI-1640, as described previously [26 (link)]. The protein concentration was measured using Coomassie Plus Protein Assay Reagent Kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions.
3
Apoptosis Pathway Protein Analysis
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Primer sequences for X-linked inhibitor of apoptosis, c inhibitor of apoptosis -1, c inhibitor of apoptosis -2, caspase-6, and caspase-7 mRNA and β-actin Western blot analysis for inhibitor of apoptosis proteins, caspase-6, and caspase-7
All the cells were lysed in lysis buffer, and the total protein concentration was estimated by Coomassie Plus Protein Assay Reagent kit (ThermoFisher, USA). Thirty microgram proteins from the cell lysate were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane film by semi-dry blotting method (Merck, New Jersey, USA) probed with respect to the primary antibodies for 15 h at 4°C. Horseradish peroxidase secondary antibodies were added and the membrane was stripped after 3 h of an incubation at 25°C. Bands were estimated in ChemiDoc XRS + by utilizing Image Lab programming (Bio-Rad) and standardized with β-actin (1:5000).
All the cells were lysed in lysis buffer, and the total protein concentration was estimated by Coomassie Plus Protein Assay Reagent kit (ThermoFisher, USA). Thirty microgram proteins from the cell lysate were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane film by semi-dry blotting method (Merck, New Jersey, USA) probed with respect to the primary antibodies for 15 h at 4°C. Horseradish peroxidase secondary antibodies were added and the membrane was stripped after 3 h of an incubation at 25°C. Bands were estimated in ChemiDoc XRS + by utilizing Image Lab programming (Bio-Rad) and standardized with β-actin (1:5000).
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