Protein assay reagent kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Protein Assay Reagent Kit is a laboratory tool used for the quantitative determination of protein concentration in solution. It provides a standardized method to measure the total protein content in a sample, enabling researchers to accurately analyze and compare protein levels across various experiments and applications.

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Protein assay reagent (28 citations) Protein assay (17 citations)

7 protocols using protein assay reagent kit

Podocyte Nuclear Protein Extraction and Western Blotting

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After subjecting to different experimental conditions, podocytes were washed twice by cold PBS, and then RIPA buffer was added for lysing. The nuclear protein was extracted from podocytes using the Nuclear and Cytoplasmic Protein Extraction Kit (Nanjing Key GEN Biotech, China). The protein assay reagent kit (Invitrogen, Waltham, MA) was used to evaluate the protein concentration. An equal amount of protein was separated on 7.5% SDS-PAGE gels electrophoresis, followed by transfer to PVDF membranes (Millipore, USA). The membranes were incubated antibodies overnight at 4 °C after blocking with 5% non-fat dry milk for at least 1 h. The primary antibodies used were as follows: rabbit anti-GAP-43 (Abcam, 1:500), rabbit anti-NFATc1 (Abcam, 1:1000), rabbit anti-histone (Cell Signaling Technology, 1:1000), rabbit anti-nephrin (Abcam, 1:2000), rabbit anti-β-actin (Affinity, 1:10000), rabbit anti-calcineurin (Abcam, 1:2000), rabbit anti-Bax (Abcam, 1:1000), and rabbit anti-Bcl-2 (Abcam, 1:1000). The next day, the anti-rabbit IgG (Cell Signaling Technology, 1:5000) was incubated for 1 h at 37 °C. Membranes were visualized using ECL Western Blotting Detection Reagents (Advansta, USA). β-actin or histone as the internal control.

Lian Z., Ke G., Zhang H., Dou C., Chen X., Li B., Zhang F., Wen S., Wu Q., Xia Y., Jiang N., Li Z., Li S., Zhao X., Ma J., Lin T., Wen F., Xu L., Li Z., Liang H., Dong W., Chen Y., Li R., Ye Z., Wang W., Liang X., Shi W., Zhang L, & Liu S. (2021). GAP-43 ameliorates Podocyte injury by decreasing nuclear NFATc1 expression. Biochemistry and Biophysics Reports, 28, 101145.

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Kidney Protein Extraction and Western Blot

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Protein extraction from the kidney cortex or cultured podocytes under different experimental conditions was conducted as previously described [22 (link)]. The nuclear protein is isolated and prepared as described in the Nuclear and Cytoplasmic Protein Extraction Kit (Nanjing KeyGEN Biotech, Nanjing, China). According to the manufacturer’s protocol, the protein concentration was evaluated using a protein assay reagent kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Equal amount of proteins was separated on 9% sodium dodecyl sulfate–polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% non-fat dry milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-ATF3 (Abcam, Cambridge, MA), rabbit anti-NFATc1(Abcam), rabbit anti-Histone (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Bax (Santa Cruz, Dallas, TX, USA), rabbit anti-Bcl-2 (Cell Signaling Technology), rabbit anti-GAPDH (Bioworld Technology, Nanjing, China), and rabbit anti-Histone (Cell Signaling Technology). Finally, membranes were detected using ECL Western Blotting Detection Reagents (Advansta, Menio Park, CA, USA).

Zhang H., Liang S., Du Y., Li R., He C., Wang W., Liu S., Ye Z., Liang X., Shi W, & Zhang B. (2017). Inducible ATF3–NFAT axis aggravates podocyte injury. Journal of Molecular Medicine (Berlin, Germany), 96(1), 53-64.

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Tyrosinase Activity Assay in B16F10 Cells

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Tyrosinase activity in B16F10 cells was assayed as described previously with modifications. Cells were washed using cold PBS and lysed with 1% sodium deoxycholate and 0.5% Triton X-100 for 2 h. Next, 150 μL of a reaction mixture containing solutions A, B, and C (2:1:1) (solution A, 2% N,N-dimethylformamide in 100 mM sodium phosphate (pH 7.1); solution B, 5 mM l-DOPA in 100 mM sodium phosphate (pH 7.1); solution C, 20 mM MBTH in H₂O) was added to the cells, followed by incubation at 37 °C for 10 min. Oxidation of l-DOPA to dopachrome was measured at 505 nm (ref. 490 nm [37 (link)]) by the microplate spectrophotometer. Some of the lysate was applied to protein assays using a protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). The protein concentration of the samples was determined by a standard curve of bovine serum albumin. The tyrosinase activity of each sample was normalized to the protein content and calculated as the relative fold induction of the treatment group to the control.

Kim H.J., Kim I.S., Dong Y., Lee I.S., Kim J.S., Kim J.S., Woo J.T, & Cha B.Y. (2015). Melanogenesis-Inducing Effect of Cirsimaritin through Increases in Microphthalmia-Associated Transcription Factor and Tyrosinase Expression. International Journal of Molecular Sciences, 16(4), 8772-8788.

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Quantitative Western Blot Analysis of Fibrotic Markers

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According to the previous description (20 (link)), Western blotting assay was performed. Total protein was extracted in radio immunoprecipitation assay (RIPA) lysis buffer (Solarbio Life Sciences) with proteinase inhibitors and phosphatase inhibitors. In order to determine the protein concentration in each lysate, we used a protein assay reagent kit (Thermo Fisher Scientific). Transfection of the proteins onto polyvinylidene difluoride membranes was carried out after electrophoresis (Millipore). Then, the membranes were blocked for 1.5 h in tris-buffered saline (TBS) with 5.0% bovine serum albumin (BSA) and probed with the primary antibodies. After being washed three times with tris buffered saline with tween 20 (TBST), the protein content was incubated with Goat Anti-Rabbit IgG (H+L), Horseradish Peroxidase (HRP) conjugate (Proteintech), or Goat Anti-Mouse IgG (H+L), HRP conjugate. The primary antibodies used were listed below: GAPDH (1:1,000, Proteintech), S100A4 (1:1,000, Abcam), and fibroblast activation protein (FAP) (1:1,000, Abcam).

Sang Q., Dai W., Yu J., Chen Y., Fan Z., Liu J., Li F., Li J., Wu X., Hou J., Yu B., Feng H., Zhu Z.G., Su L., Li Y.Y, & Liu B. (2022). Identification of prognostic gene expression signatures based on the tumor microenvironment characterization of gastric cancer. Frontiers in Immunology, 13, 983632.

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Cytotoxicity Protein Assessment in LO2 Cells

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For cytotoxicity assessment, total proteins from LO2 cells treated with PIX (16~32 μg/mL) for 24 h were extracted using a Protein Assay reagent kit (Thermo Fisher Scientific). Then, the samples were separated by 10% SDS-PAGE electrophoresis. Subsequently, the proteins were transferred onto PVDF membranes and blocked with 5% skim milk. The membrane was incubated with the primary antibodies anti-β-actin and anti-β-tubulin, followed by HRP-conjugated secondary antibodies (Abcam). Protein bands were developed and analyzed with Alpha Imager 2200 software (Alpha Innotech Corporation).

She P., Li Z., Li Y., Liu S., Li L., Yang Y., Zhou L, & Wu Y. (2022). Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin. Microbiology Spectrum, 10(6), e02114-22.

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Micro BCA Protein Quantification

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Total protein determinations were performed on BAL samples using the Micro BCA (bicinchoninic acid) protein assay reagent kit (Pierce Biotechnology, USA) according to the manufacturer’s instructions. In brief, each sample was diluted 1:20 with the Working Reagent, incubated at 37°C for 30 minutes before reading in a spectrophotometer at 562 nm. Values were compared to that for a freshly prepared protein standard curve.

Hashemian S.M., Mortaz E., Tabarsi P., Jamaati H., Maghsoomi Z., Khosravi A., Garssen J., Masjedi M.R., Velayati A.A., Folkerts G., Barnes P.J, & Adcock I.M. (2014). Elevated CXCL-8 expression in bronchoalveolar lavage correlates with disease severity in patients with acute respiratory distress syndrome resulting from tuberculosis. Journal of Inflammation (London, England), 11, 21.

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Quantifying Protein Levels via BCA Assay

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BCA (bicinchoninic acid) protein assay reagent kit (Pierce Biotechnology, Rockford, IL, USA) was used. Detailed information is shown in Additional file 1: Supplement-Methods.

Xu X., Jia C., Luo S., Li Y., Xiao F., Dai H, & Wang C. (2017). Effect of HA330 resin-directed hemoadsorption on a porcine acute respiratory distress syndrome model. Annals of Intensive Care, 7, 84.

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