Bca assay

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The BCA assay is a colorimetric detection method used to quantify the total protein concentration in a sample. It involves the reduction of copper(II) to copper(I) by protein in an alkaline medium, which then chelates 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 in the sample.

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BCA protein assay kit (29 citations) BCA assay kit (6 citations) BCA protein assay (5 citations)

7 protocols using bca assay

Protein Extraction and Western Blotting

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Cells were lysed with M-PER^® Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with 1X protease inhibitor cocktail (Roche), 0.2 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride (PMSF). BCA assay (Santa Cruz) was used for protein quantification. Cell lysates or IP elutes were subjected to SDS/PAGE followed by either conventional wet transfer or dry transfer using iBlot^® Gel Transfer Device (Invitrogen). Membranes were incubated with antibodies as indicated and exposed to secondary HRP-conjugated antibodies (Millipore).

Lin D.C., Xu L., Chen Y., Yan H., Hazawa M., Doan N., Said J.W., Ding L.W., Liu L.Z., Yang H., Yu S., Kahn M., Yin D, & Koeffler H.P. (2015). Genomic and functional analysis of the E3 ligase PARK2 in glioma. Cancer research, 75(9), 1815-1827.

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Quantification of FSIP1 Protein Levels

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Total proteins were extracted using a protein extraction kit (ProMab, USA) followed by centrifugation. Protein concentration was quantified using BCA assay (Santa Cruz Biotechnology, USA), individual cell lysate (30 μg/lane) was then separated on sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. Membrane was blocked with 5% fat-free dry-milk in TBST and incubated with rabbit anti-FSIP1 antibody (1:100 dilution; Abcam, USA) followed by rabbit anti- β-actin antibody (1:5000 dilution; Abcam) at 4°C overnight. Bound antibodies were detected with HRP-conjugated anti-rabbit, anti-mouse, or anti-goat immunoglobulin G (IgG) at room temperature for 1 h and visualized with enhanced chemiluminescence (Santa Cruz Biotechnology, USA). Purified mouse, rabbit, or goat IgG was used as a negative control. Relative levels of targeting protein to the control β-actin were determined using ImmuNe software.

Zhang H., Luo M., Jin Z., Wang D., Sun M., Zhao X., Zhao Z., Lei H., Li M, & Liu C. (2015). Expression and clinicopathological significance of FSIP1 in breast cancer. Oncotarget, 6(12), 10658-10666.

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Protein Analysis of Rat Hippocampus Post-TBI

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Western blots of protein from fresh hippocampal tissue were obtained from rats perfused with cold ACSF (4°C) 3 days and 1 week after FPI or sham-injury as described previously (Li et al., 2015 (link)) using antibodies listed in Table 1. Protein concentration of the sample lysates was measured using BCA assay (Santa Cruz). Equal amounts of protein samples were diluted at a ratio of 1:1 in Laemmli sample buffer (Sigma) and separated on pre-cast gel (4–12% Tris–glycineBio-Rad). Chemiluminescent detection was performed with ECL western blotting detection reagent (Westdura, Thermo Scientific) using FluoroChem 8800. Densitometric quantification was determined using Image-J software (NIH) and normalized to β-actin density.

Korgaonkar A.A., Nguyen S., Li Y., Sekhar D., Subramanian D., Guevarra J., Pang K.C, & Santhakumar V. (2020). Distinct cellular mediators drive the Janus Faces of Toll-like Receptor 4 regulation of network excitability which impacts working memory performance after brain Injury. Brain, behavior, and immunity, 88, 381-395.

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Western Blot Analysis of CHCHD2 and CD105

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Liver tissues or cells were homogenized in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) and the protein concentrations were quantified by BCA assay (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's instruction. A total of 40 µg lysates were separated by SDS-PAGE and transferred to PVDF membrane, which was then blocked with 5% non-fat dry milk for 1 h. The membranes were incubated with CHCHD2 antibody (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and CD105 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, followed by incubation with horse radish peroxidase conjugated secondary antibody (1:600, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. ECL detection reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect the immunereactive bands. All bands were imaged and analyzed by densitometry scanning (Chemilmager IM5500, Alpha Innotech, USA).

Yao Y., Su J., Zhao L., Li R., Liu K, & Wang S. (2019). CHCHD2 promotes hepatocellular carcinoma and indicates poor prognosis of hepatocellular carcinoma patients. Journal of Cancer, 10(27), 6822-6828.

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Protein Expression Analysis Protocol

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Cells were lysed with lysis buffer (20 mM HEPES (pH 7.4), 350 mM sodium chloride, 1.5 mM magnesium chloride, 1 mM EGTA, 10% (v/v) glycerol, 1% Triton X-100, a mixture of protease inhibitors (Roche, Indianapolis, IN, USA), 0.2 mmol/L sodium orthovanadate and 1 mmol/L phenylmethylsulfonyl fluoride). BCA assay (Santa Cruz, Dallas, TX, USA) was used for protein quantification. Cell lysates or immunoprecipitation elutes were subjected to SDS-PAGE followed by conventional wet transfer. Membranes were incubated with antibodies as indicated and exposed to secondary horseradish peroxidase-conjugated antibodies (Millipore, Billerica, MA, USA). Antibodies used in this study were listed in Supplementary Table S2. The relative amount of p63 and ZNF750 protein was quantified by densitometry using Photoshop CS6 Extended (Adobe, San Jose, CA, USA) and normalized by GAPDH. Values from each cell line were then divided by that of UMSCC1.

Hazawa M., Lin D.C., Handral H., Xu L., Chen Y., Jiang Y.Y., Mayakonda A., Ding L.W., Meng X., Sharma A., Samuel S., Movahednia M.M., Wong R.W., Yang H., Tong C, & Koeffler H.P. (2016). ZNF750 is a lineage-specific tumour suppressor in squamous cell carcinoma. Oncogene, 36(16), 2243-2254.

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BSA Adsorption on Scaffold Powders

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The BSA protein adsorption on scaffolds’ powders was obtained according to the protocol reported by Shi et al. [19 (link)]. In brief, 10 mg of powders (CaP_Si0, CaP_Si1, CaP_Si2, CaP_Si3 and CaP_Si4) were incubated with 1 mL of bovine serum albumin (BSA, Sigma Aldrich, ≥96%) protein (250 μg/mL, protein/phosphate-buffered saline) in triplicates for 4 h at 37 °C. Following the incubation, protein concentration of the supernatant was measured using the BCA assay (Santa Cruz Biotechnology, Dallas, TX, USA). Adsorbed protein concentration was calculated after subtracting the protein concentration of the supernatant (non-adsorbed protein) from the initial BSA concentration. The results are presented as the percentage (%) of adsorbed protein in relation to the negative control.

Ressler A., Bauer L., Prebeg T., Ledinski M., Hussainova I., Urlić I., Ivanković M, & Ivanković H. (2022). PCL/Si-Doped Multi-Phase Calcium Phosphate Scaffolds Derived from Cuttlefish Bone. Materials, 15(9), 3348.

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Quantifying Epithelial-Mesenchymal Transition Markers

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The CSC were collected at different time points after transfection, and total proteins were extracted using a total protein extraction kit (ProMab). After quantification of protein concentrations using a BCA assay (Santa Cruz Biotech), the individual cell lysates (20 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free dry-milk in TBST and incubated with rabbit anti-Slug (1:500; Cell Signaling Technology), mouse anti-vimentin (1:400), mouse anti-GAPDH (1:800), goat anti-E-cadherin (1:500; Santa Cruz Biotech), mouse anti-Snail (1:500; Cell Signaling Technology), rabbit anti-N-cadherin (1:500), and rabbit anti-CK 18 (1:5000; Abcam) at 4°C overnight, respectively. After washing, the bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG at room temperature for 1 h and visualized using enhanced chemiluminescence. The relative levels of targeting proteins to the control GAPDH were determined using ImmuNe software.

Wang D., Lu P., Zhang H., Luo M., Zhang X., Wei X., Gao J., Zhao Z, & Liu C. (2014). Oct-4 and Nanog promote the epithelial-mesenchymal transition of breast cancer stem cells and are associated with poor prognosis in breast cancer patients. Oncotarget, 5(21), 10803-10815.

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