After continuous infusion for eight hours, a small piece of the left ventricular myocardium was homogenized in a cold lysis buffer supplemented with protease inhibitors, and protein concentrations were evaluated using a bicinchoninic acid protein assay kit (Elabscience, Wuhan, China) with Tubulin as loading control. The samples of tissue lysis were heated to 95 °C for five minutes. Protein samples (40 µg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride membranes overnight at 4 °C. Membranes were incubated with the appropriate primary antibody including anti-Bcl-2, anti-BAX, and anti-Tubulin, all from Elabscience, overnight at 4 °C. After washing with tris-buffered saline (TBST) three times, five minutes for each time, the secondary anti-rabbit immunoglobulin G antibody was then added and incubated for 30 minutes. After washing with TBST three times, proper amount of electrochemical luminescent substrate was added and incubated with the membranes in the dark. The results were scanned and processed by the ImageJ software.
Bicinchoninic acid protein assay kit
Manufactured by Elabscience
Sourced in China
The Bicinchoninic acid (BCA) protein assay kit is a colorimetric assay used for the quantitative determination of total protein concentration. The kit utilizes the well-known reaction between BCA and proteins, resulting in the formation of a purple-colored reaction product that can be detected spectrophotometrically.
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Lab products found in correlation
2 protocols using bicinchoninic acid protein assay kit
1
Protein Expression Analysis of Cardiac Myocardium
2
Western Blot Analysis of CD4+ T Cells
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CD4 + T cells were collected at 3 days after incubation and lysed with lysis buffer on ice. The protein concentrations were measured using a bicinchoninic acid protein assay kit (Elabscience, Wuhan, China). Proteins (40 μg) were separated on a 10% sodium dodecyl sulfate gel and transferred to a polyvinylidene fluoride membrane, followed by blocking with 5% skim milk for 1 h. The membrane was then incubated overnight at 4°C with primary antibody (Affinity Biosciences, Cincinnati, OH, USA) to rat GAPDH (1:1,000), PTPN2 (1:1,000), STAT3 (1:1,000), or p-STAT3 (1:1,000), followed by three washes with tris-buffered saline containing 0.1% Tween 20 (TBST). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; Elabscience) for 1 h at room temperature. After an additional three washes with TBST, the protein bands were visualized using an enhanced chemiluminescence reagent and analyzed using BandScan software (Glyko, Novato, CA, USA).
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