To extract the total protein from each sample, the PRO-PREPTM kit (American Intron Biotechnology) was used. After, the total amount of each protein was quantified under the guidelines of Bradford Protein Analysis (Bio-Rad, CA, USA) and protein samples were kept at −80°C until they were used in the analysis. 30 μg of total protein extracted from each sample was separated on a 13% sodium dodecyl sulphate-polyacrylamide gel and transferred to a Immunoblot polyvinylidene fluoride membranes (Bio-Rad, USA). It was then detected using secondary antibody (anti-rabbit and/or anti-mouse secondary antibody; diluted 1:5,000; Abcam, MA) after inducing antigen antibody reactions using MC1R (diluted 1:1,000; TA308794, OriGene, USA, MD), TYR (diluted 1:1,000; AB6211, Abcam, UK, Cambridge) and β-actin (diluted 1:5,000; AB49900, Abcam) in the membrane where the protein was transferred. The membrane was then fluorescently reacted with ECL and analyzed after 1–5 min of exposure in diagnostic films.
Bradford protein analysis
Manufactured by Bio-Rad
Sourced in United States
The Bradford protein analysis is a colorimetric assay used to quantify the total protein concentration in a sample. It is based on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be measured spectrophotometrically. The assay provides a rapid and simple method for determining protein levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Immunoblot polyvinylidene fluoride membranes, by Bio-Rad (1 mentions)
Ab6211, by Abcam (1 mentions)
Ab49900, by Abcam (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Iblot2 pvdf stack, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail 100, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
4 protocols using bradford protein analysis
1
Protein Extraction and Quantification
2
Bradford Protein Analysis of Placental and Mitochondrial Extracts
3
Protein Extraction and Western Blotting
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The cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Danvers, MA, USA) containing the Halt Protease and Phosphatase Inhibitor Cocktail (100×) (Thermo Fisher Scientific, Rockford, IL, USA), and the concentration of protein was calculated using Bradford protein analysis (Bio-Rad, Hercules, CA, USA). For Western blotting, protein samples (20 µg) were dissolved in 4× lithium dodecyl sulfate sample buffer and 10× reducing sample agent (Invitrogen), heated at 95°C for 10 minutes, and loaded on a 4% to 12% Bis-Tris NuPAGE gel (Thermo Fisher Scientific). The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using the iBlot2 PVDF stack (Invitrogen), and the membranes were blocked with 5% bovine serum albumin in tris-buffered saline with Tween-20 (TBST) for 1 hour at room temperature, following which they were incubated at 4°C overnight with the following specific primary antibodies (Supplementary Table 2 ). After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized by enhanced chemiluminescence reagent (DAWINBio, Hanam, Korea).
4
Chrysin Modulates Apoptosis Markers
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Western blotting was performed to analyze the expression of apoptosis-related proteins. MC-3 oral cancer cells were cultured in a 75 cm2 flask at 1 × 106 cells/mL for 24 h and then treated with chrysin (0, 50, and 100 µM). After 24 h, cells were harvested using trypsin-EDTA. A cell lysis buffer was added to the harvested cells for 20 min at 4 °C, and the mixture was centrifuged (15,920× g, 5 min, 4 °C) to obtain the supernatant as the cell lysate. Bradford protein analysis (Bio-Rad Laboratories) was used to quantify the concentration of the extracted proteins. The cell lysate was separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to nitrocellulose membranes (Hercules, CA, USA). After 1 h blocking of the membranes in 5% skim milk-TBST (20 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), the target primary antibody (1:1000) was added for an overnight (4 °C) reaction. Next, rabbit IgG or mouse IgG (1:2000) was added for a 1 h reaction (22–23 °C). Afterwards, ECL detection reagents were used for blotting, while density was measured using Image J Launcher (provided by NCBI).
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