Bicinchoninic acid protein assay

Manufactured by Boster Bio

Sourced in China

The Bicinchoninic acid (BCA) protein assay is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. It involves the reduction of copper(II) ions to copper(I) ions by proteins in an alkaline medium, followed by the chelation of the copper(I) ions with bicinchoninic acid to produce a purple-colored complex that absorbs light at 562 nm. The intensity of the color is proportional to the protein concentration.

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Bicinchoninic acid assay (6 citations)

4 protocols using bicinchoninic acid protein assay

Protein Expression Analysis by Western Blot

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Total protein was extracted from cells using enhanced radioimmunoprecipitation assay lysis buffer (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) containing protease inhibitor, and the concentration was determined using a bicinchoninic acid protein assay (Boster). The protein was then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto polyvinylidene fluoride membranes. The membrane was blocked with 5% bovine serum albumin for 1 h at room temperature and probed at 4°C overnight with the following diluted primary antibodies (Abcam): HO-1 (ab68477, 1:10,000, Rabbit), Occludin (ab216327, 1:1,000, Rabbit), SP1 (ab227383, 1:2,000, Rabbit), HDAC4 (ab235583, 1:1,000, Rabbit), HMGB1 (ab18256, 1:500, Rabbit), Acetyl-Lys (ab190479, 1:2,000, Rabbit), GAPDH (ab181603, 1:5,000, Rabbit), and Lamin A (ab133256, 1:10,000, Rabbit). After 3 washes using Tris-buffered saline Tween-20, the membrane was reprobed with the horseradish peroxidase-labeled secondary antibody (goat anti-rabbit, ab205718, 1:1,000; goat anti-mouse, ab205719, 1:1,000; Abcam) for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence (Baoman Biotechnology Co., Ltd., Shanghai, China). With GAPDH and Lamin A as internal references, ImageJ software was used to analyze the gray value.

Liu Z.M., Wang X., Li C.X., Liu X.Y., Guo X.J., Li Y., Chen Y.L., Ye H.X, & Chen H.S. (2022). SP1 Promotes HDAC4 Expression and Inhibits HMGB1 Expression to Reduce Intestinal Barrier Dysfunction, Oxidative Stress, and Inflammatory Response after Sepsis. Journal of Innate Immunity, 14(4), 366-379.

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Cytokine Profiling in Rat Choroid Plexus

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Rats were anesthetized with pentobarbital (40 mg/kg intraperitoneal). CSF was collected through the cisterna magna; then, the rats were decapitated to obtain the choroid plexus. The CSF was centrifuged at 10,000 rpm to separate the supernatant. The choroid plexus was homogenized in ice-cold phosphate-buffered saline with protease inhibitor cocktails (Sigma-Aldrich, USA). Total protein concentration was quantified using a bicinchoninic acid protein assay (Boster, China). The CSF and choroid plexus cytokine levels were measured using inflammation cytokine assay kits (RayBiotech, USA) according to the manufacturer’s instructions.

Zhang Z., Tan Q., Guo P., Huang S., Jia Z., Liu X., Feng H, & Chen Y. (2022). NLRP3 inflammasome-mediated choroid plexus hypersecretion contributes to hydrocephalus after intraventricular hemorrhage via phosphorylated NKCC1 channels. Journal of Neuroinflammation, 19, 163.

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Western Blot Analysis of SIRT1 and MMPs

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Cells specimens were lysed with RIPA lysis buffer (Boster). Protein concentrations were measured by bicinchoninic acid protein assay (Boster). Equal amounts of total protein were boiled in sample buffer and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Proteins were then transferred to a polyvinylidene fluoride membrane (Millipore) and blocked with 5% skim milk powder at room temperature for 1 hour. The membrane was incubated with rabbit monoclonal antibody against SIRT1 (#2496; Cell Signaling Technology), matrix metalloproteinase ( MMP)-2 (#40994; Cell Signaling Technology), MMP-9 (#13667; Cell Signaling Technology), and goat anti-rabbit immunoglobulin G. An electrochemiluminescence kit was used to visualize the protein bands. Protein levels were calculated relative to β-actin.

Liu Y., Li Q., Liang H., Xiang M., Tang D., Huang M., Tao Y., Ren M., Zhao M., Wang J., Shu L., He Z., Wang F, & Li Y. (2020). MiR-34a Regulates Nasopharyngeal Carcinoma Radiosensitivity by Targeting SIRT1. Technology in Cancer Research & Treatment, 19, 1533033820940424.

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Western Blot Analysis of Liver Proteins

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Liver tissue was homogenized in RIPA Lysis Buffer containing PMSF (a protease inhibitor (Beyotime Institute of Biotechnology), and total protein concentrations were determined by bicinchoninic acid protein assay (Boster). About 50 micrograms of protein were loaded on 5–10% gradient gels. Proteins were transferred to a PVDF membrane(Thermo Scientific) for 70 min at 100 V. The membranes were incubated in blocking buffer for 2 hours before the addition of primary antibody including Wnt5a (Abcam, USA), JNK1 (Santa Cruz Biotechnology, USA), NF-κB p65 (Abcam, USA) and COX-2 (Proteintech Group, PTG, USA). All of the primary antibodies were used at a 1∶400 dilution. All secondary antibodies were diluted 1∶1000 except for anti-NF-κB p65, which was diluted 1∶3000. Proteins were detected via enhanced chemiluminescence (Beyotime Institute of Biotechnology), and the intensity of protein bands were quantified using the Quantity One software (Bio-Rad, USA).

Tian F., Zhang Y.J., Li Y, & Xie Y. (2014). Celecoxib Ameliorates Non-Alcoholic Steatohepatitis in Type 2 Diabetic Rats via Suppression of the Non-Canonical Wnt Signaling Pathway Expression. PLoS ONE, 9(1), e83819.

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