Bca protein quantitation assay

Manufactured by Keygen Biotech

Sourced in China

The BCA Protein Quantitation Assay is a colorimetric detection kit used for the quantification of total protein concentration in a sample. The assay utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ to Cu+ in an alkaline environment, and the resulting Cu+ ions chelate with BCA to produce a purple-colored complex that can be measured spectrophotometrically.

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Lab products found in correlation

Whole cell lysis assay, by Keygen Biotech (3 mentions) Pvdf membrane, by Merck Group (2 mentions) Chemiluminescent detection kit, by Merck Group (2 mentions) Whole cell protein extraction kit, by Keygen Biotech (2 mentions) Enhanced chemi luminescence (ecl), by Merck Group (2 mentions) Ab108357, by Abcam (2 mentions) Ab32147, by Abcam (2 mentions) Ecl reagent, by Merck Group (2 mentions) Hrp conjugated secondary antibody, by Merck Group (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Immobilon western chemiluminescent horseradish peroxidase substrate, by Merck Group (1 mentions) Protease inhibitor, by Keygen Biotech (1 mentions) Il 1β, by Proteintech (1 mentions) Amersham imager 600, by Cytiva (1 mentions) Ripa lysis buffer, by Keygen Biotech (1 mentions) Amersham imager, by Cytiva (1 mentions) Ab32053, by Abcam (1 mentions) Ab133327, by Abcam (1 mentions) Ab53699, by Abcam (1 mentions) Vimentin, by Abcam (1 mentions)

Close spelling variants of this product

BCA assay (29 citations) BCA protein assay (20 citations) ) protein assay (2 citations)

19 protocols using bca protein quantitation assay

Quantifying Intracellular Carotenoids and Starches

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Intracellular carotenoids were analyzed according to a previous method with minor modifications (Sun et al., 2019 (link)). Briefly, 20 mg freeze-dried algae powder was ground using a mortar and pestle in liquid nitrogen and then extracted three times with acetone. The extraction system was allowed to stand in an icebox for 5 min. Then, the extract was dried in a vacuum concentrator after centrifugation. When the extract completely volatilized, 100 μL acetone was added to dissolve the carotenoids. The supernatant was collected for detection by an Agilent 1260 series binary HPLC system.
Intracellular starches were determined as described in our previous study (Diao et al., 2019 (link)). For intracellular proteins, 5 mg freeze-dried algae powders were used to extract total proteins with an Algae Protein Extraction Kit (BestBio, China) according to the manufacturer’s instructions. Similarly, the total protein concentration was determined by the BCA Protein Quantitation Assay (KeyGEN BioTECH, China) according to the manufacturer’s instructions.

Liu L., Diao J., Bi Y., Zeng L., Wang F., Chen L, & Zhang W. (2022). Rewiring the Metabolic Network to Increase Docosahexaenoic Acid Productivity in Crypthecodinium cohnii by Fermentation Supernatant-Based Adaptive Laboratory Evolution. Frontiers in Microbiology, 13, 824189.

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Quantifying Protein Expression via Western Blot

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Western blot experiments were performed to determine the protein expression in cell lysates. The protein concentration was quanti ed with BCA protein quantitation assay (KeyGen Biotech, China). Equal protein amounts (30 μg) were loaded onto SDS-PAGE gels and transferred to PVDF membranes (Millipore Sigma, China). Phosphorylated protein or common protein was blocked for 2 h at room temperature in 5% BSA or 5% skim milk in TBS with 0.05% Tween-20. Protein bands were probed overnight with the suitable primary antibody at 4°C. Proteins were visualized using HRP-conjugated secondary antibody and a chemiluminescent detection kit (Millipore, USA). The amount of target protein was calculated by grey scanning.

Zhang X., Ye L., Xu H., Zhou Q., Tan B., Yi Q., Yan L., Xie M., Zhang Y., Tian J., & Zhu J. (2021). Nrf2 is required for structural and metabolic maturation of human induced pluripotent stem cell-derived cardiomyocytes.

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Western Blot Analysis of Cellular Proteins

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For Western blot analysis, total proteins from cell lines and fresh-frozen tissues were extracted using the Whole Cell Protein Extraction Kit (KeyGEN BioTECH, Wuhan, China). A BCA Protein Quantitation Assay (KeyGEN BioTECH) was used to measure the protein concentration. An equal amount (30 μg) of protein sample was separated on 10% sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, MA). The membranes then were blocked with 5% nonfat dry milk in Tris-buffered saline/0.1% Tween 20 for 1 hour at room temperature. The membranes were incubated with appropriate primary antibodies (Table 3) overnight at 4°C. The next day, the membranes were washed and incubated with horseradish-peroxidase–conjugated secondary antibody. Proteins were visualized using Immobilon Western Chemiluminescent Horseradish-Peroxidase Substrate (Millipore).

Cai Q., Shi P., Yuan Y., Peng J., Ou X., Zhou W., Li J., Su T., Lin L., Cai S., He Y, & Xu J. (2020). Inflammation-Associated Senescence Promotes Helicobacter pylori–Induced Atrophic Gastritis. Cellular and Molecular Gastroenterology and Hepatology, 11(3), 857-880.

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Protein Expression Analysis After Ischemic Injury

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Following reperfusion, ipsilateral brain tissue was homogenized in RIPA lysis buffer containing protease inhibitors (Nanjing KeyGen Biotech Co., Ltd.). The protein concentration of the supernatants (24,148.8 × g, 5 min, 4°C) was measured using a BCA Protein Quantitation Assay (Nanjing KeyGen Biotech Co., Ltd.). Equal amounts of protein (30 μg) were loaded onto 8-10% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoresis, bands were transferred onto PVDF membranes. After being blocked with 5% BSA (OriGene Technologies, Inc.) for 2 h at 37°C, the membranes were incubated overnight at 4°C using the aforementioned primary antibodies against claudin-5, ZO-1, occludin, laminin and collagen IV or TNF-α (1:1,000; cat. no. 17590-1-AP; ProteinTech Group, Inc.), IL-1β (1:500; cat. no. ab200478; Abcam) or IL-6 (1:2,000; cat. no. 66146-1-Ig; ProteinTech Group, Inc.). The membranes were washed and incubated with HRP-conjugated secondary antibody (1:2,000; cat. no. SA00013-2; ProteinTech Group, Inc.) for 2 h at room temperature. Visualization was performed using ECL kit (cat. no. 34577; Thermo Fisher Scientific, Inc.). The immunoblots were visualized using a computerized image analysis system (Amersham Imager 600; Cytiva), and the results were expressed as the ratio of corresponding protein to β-actin (1:5,000; cat. no. SA00001-7L; ProteinTech Group, Inc.).

Guo Y., Dong L., Gong A., Zhang J., Jing L., Ding T., Li P.A, & Zhang J.Z. (2021). Damage to the blood-brain barrier and activation of neuroinflammation by focal cerebral ischemia under hyperglycemic condition. International Journal of Molecular Medicine, 48(1), 142.

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Quantitative Protein Expression Analysis

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Western blot experiments were performed to determine the protein expression in cell lysates. The protein concentration was quantified with BCA protein quantitation assay (KeyGen Biotech, China). Equal protein amounts (30 μg) were loaded onto SDS-PAGE gels and transferred to PVDF membranes (Millipore Sigma, China). Phosphorylated protein or common protein was blocked for 2 h at room temperature in 5% BSA or 5% skim milk in TBS with 0.05% Tween-20. Protein bands were probed overnight with the suitable primary antibody at 4 °C. Proteins were visualized using HRP-conjugated secondary antibody and a chemiluminescent detection kit (Millipore, USA). The amount of target protein was calculated by gray scanning.

Zhang X., Ye L., Xu H., Zhou Q., Tan B., Yi Q., Yan L., Xie M., Zhang Y., Tian J, & Zhu J. (2021). NRF2 is required for structural and metabolic maturation of human induced pluripotent stem cell-derived ardiomyocytes. Stem Cell Research & Therapy, 12, 208.

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Western Blot Analysis of Cell Cycle Regulators

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The cells were washed twice with 4°C PBS and then lysed in cold RIPA buffer with protease inhibitors. The BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China) was used to measure the protein concentration. The total protein was transferred to a nitrocellulose membrane after denaturing by 10% SDS‐PAGE. The membranes were blocked with 5% non‐fat milk in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C (dilution ratio 1:2000). The membranes were washed three times with TBST and then incubated with secondary antibodies (anti‐rabbit IgG or anti‐mouse IgG) for 1 h at room temperature. The membranes were washed three times with TBST, and then, the targeted proteins were detected by the ECL (EMD Millipore, MA, USA) method. XPOT, CDK1, CDK2, CDK4, CCNA1, CCNB1, CCNB2, CCNE2 antibodies for western blot were obtained from Bioss (bs‐14673R), Abcam (ab133327), Abcam (ab32147), Abcam (ab108357), Abcam (ab53699), Abcam (ab32053), Abcam (18250), Abcam (40890).

Lin J., Hou Y., Huang S., Wang Z., Sun C., Wang Z., He X., Tam N.L., Wu C, & Wu L. (2018). Exportin‐T promotes tumor proliferation and invasion in hepatocellular carcinoma. Molecular Carcinogenesis, 58(2), 293-304.

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Western Blot Analysis for EMT Markers

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Cells were lysed in cold radioimmunoprecipitation assay buffer containing protease inhibitors. Protein concentrations were measured by a BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China). Total protein was transferred to a nitrocellulose membrane after denaturation in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. After incubation with primary antibodies and secondary antibodies, the targeted proteins were detected using ECL reagents (EMD Millipore, MA). Primary antibodies against E‐cadherin (1:1000; Abcam, Cambridge, MA), vimentin (1:1000; Abcam) and N‐cadherin (1:1000; Abcam) were used.

Sun C., Huang S., Wang H., Xie R., Zhang L., Zhou Q., He X, & Ju W. (2019). Non‐SMC condensin I complex subunit H enhances proliferation, migration, and invasion of hepatocellular carcinoma. Molecular Carcinogenesis, 58(12), 2266-2275.

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Western Blot Analysis of Liver Proteins

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Whole liver protein was extracted from rat liver using lysis buffer (KeyGen Biotech, Nanjing, China) containing protease and phosphatase inhibitors. Protein concentration was determined by a BCA protein quantitation assay (KeyGen Biotech, Nanjing, China). Briefly, 20 μg of proteins were subjected to 8%–12% SDS-PAGE and then transferred onto PVDF membranes. Blots were blocked with blocking buffer for 2 h and then incubated with appropriate primary antibodies at 4°C overnight. Antibodies against GAPDH, PCNA, AKT, p-AKT, Bcl2, Bcl-xl, STAT3, p-STAT3, FASN, and Cyclin D1 were purchased from Abcam. Antibodies against mTOR, p-mTOR, 4EBP1, p-4EBP1, GSK3β, p-GSK3β, FOXO1, YAP, p70 S6K, p-p70 S6K, and SOCS3 were purchased from Cell Signaling Technology. Antibodies against β-catenin and SREBP1c were purchased from Santa Cruz Biotechnologies. After washing 3 times with Tris-buffered saline containing 1% Tween 20 (TBST), the membranes were incubated with the secondary antibody goat anti-rabbit IgG and goat anti-mouse IgG (Abcam, UK) at room temperature for 2 hours. After washing 3 times with TBST, signals of the target protein were developed using the enhanced chemiluminescence substrates (ECL, Thermo Fisher Scientific).

Liu Y., Yang F., Li J., Wang J., Wang X., Zhang Y., Yuan X., Zhu W, & Shi X. (2018). Mesenchymal Stem Cells Enhance Liver Regeneration via Improving Lipid Accumulation and Hippo Signaling. Stem Cells International, 2018, 7652359.

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Western Blot Protein Detection Protocol

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The tissues and cells were washed twice with 4°C PBS and then lysed in cold RIPA buffer with protease inhibitors. The BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China) was used to measure the protein concentration. The total protein was transferred to a nitrocellulose membrane after denaturing by 10% SDS-PAGE. The membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with the primary antibodies overnight at 4°C (dilution ratio 1:2,000). The membranes were washed three times with TBST and then incubated with secondary antibodies (anti-rabbit IgG or anti-mouse IgG) for 1 h at room temperature. The membranes were washed three times with TBST, and then, the targeted proteins were detected by the ECL reagent (EMD Millipore, MA, USA) method.

Huang S., Zhang C., Sun C., Hou Y., Zhang Y., Tam N.L., Wang Z., Yu J., Huang B., Zhuang H., Zhou Z., Ma Z., Sun Z., He X., Zhou Q., Hou B, & Wu L. (2020). Obg-like ATPase 1 (OLA1) overexpression predicts poor prognosis and promotes tumor progression by regulating P21/CDK2 in hepatocellular carcinoma. Aging (Albany NY), 12(3), 3025-3041.

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Western Blot Analysis of Cell Signaling

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PBS at 4 °C was used to wash the cells twice. Thereafter, protease inhibitors in cold RIPA buffer were used to lyse the cells. Protein concentration was determined using BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China). Total protein of the cells were denatured using 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) was used to block the membrane for 1 h at room temperature. The membranes were then incubated with the primary antibodies overnight at 4 °C (dilution ratio 1:2000). TBST was used to wash the membranes three times and they were subsequently incubated with secondary antibodies (anti-rabbit IgG or anti-mouse IgG) for 1 h at room temperature. TBST was once again used to wash the membranes three times. The targeted proteins were identified using the ECL (EMD Millipore, MA, USA) method. SKA3, CCNE2, CCNA2, CDK4, CDK2, P53, P53-pSer15, P53-pSer46 antibodies used for western blot in this research were purchased from Bioss (bs-7848R), Abcam (ab32147), Abcam (ab108357), Abcam (ab181591), Abcam (ab32103), Abcam (ab32389), CST #9284 and CST#2521.

Hou Y., Wang Z., Huang S., Sun C., Zhao J., Shi J., Li Z., Wang Z., He X., Tam N.L, & Wu L. (2019). SKA3 Promotes tumor growth by regulating CDK2/P53 phosphorylation in hepatocellular carcinoma. Cell Death & Disease, 10(12), 929.

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