Bca protein assay reagent

Manufactured by Merck Group

Sourced in United States

The BCA protein assay reagent is a colorimetric detection kit used for the quantitative determination of protein concentration in a sample. The reagent utilizes the bicinchoninic acid (BCA) method to produce a purple-colored reaction product, the intensity of which is proportional to the protein concentration in the sample.

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Lab products found in correlation

Mini protean 3 system, by Bio-Rad (1 mentions) Odyssey imaging system, by LI COR (1 mentions) Nitrocellulose blotting membrane, by GE Healthcare (1 mentions) Nupage novex 10 bis tris protein gel, by Thermo Fisher Scientific (1 mentions) Image studio lite ver 5, by LI COR (1 mentions) Phospho akt ser473, by Cell Signaling Technology (1 mentions) Phospho akt thr308, by Cell Signaling Technology (1 mentions) Nupage lds sample buffer 4x, by Thermo Fisher Scientific (1 mentions) Mini trans blot, by Bio-Rad (1 mentions) Pi3 kinase p110α, by Cell Signaling Technology (1 mentions) Coomassie brilliant blue staining, by Bio-Rad (1 mentions) Ripa cell lysis buffer, by Thermo Fisher Scientific (1 mentions) Enhanced chemiluminescence detection system, by Cytiva (1 mentions) Tnf α and il 6, by BD (1 mentions) 96 well plate, by SPL Life Sciences (1 mentions) 2 2 azinobis, by BD (1 mentions) Automated microplate elisa reader, by Molecular Devices (1 mentions) Triton x 100, by Merck Group (1 mentions) Hybond ecl nitrocellulose membrane, by GE Healthcare (1 mentions) Porcine trypsin, by Promega (1 mentions)

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BCA assay (242 citations) BCA protein assay (99 citations) Protein assay reagents (12 citations) BCA reagent (7 citations) Protein assay (6 citations) BCA protein reagent (3 citations)

10 protocols using bca protein assay reagent

Western Blot Analysis of Akt Pathway

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To ensure equal sample loading, protein concentration was determined using bicinchoninic acid (BCA) protein assay reagent (Sigma, UK) and adjusted accordingly. NuPAGE LDS Sample Buffer (4X) (Thermofisher Scientific, UK) plus 5% β-mercaptoethanol were added to the samples, which were subsequently denatured by heating to 100 °C for 10 min. Samples were then loaded on NuPAGE Novex 10% Bis–Tris protein gels (Thermofisher scientific, UK) using the Mini Protean III system (Bio-Rad, UK). Proteins were transferred onto nitrocellulose blotting membrane (GE Healthcare Life Sciences, UK) through wet transfer in a Bio-Rad Mini Trans-Blot. The membranes were blocked for 1 h using 5% bovine serum albumin/PBS tween and subsequently incubated with appropriate primary antibodies at 4 °C overnight.
The following primary antibodies being used were acquired from Cell Signalling Technology: Akt (#9272), Phospho-Akt (Ser473) (#9271), Phospho-Akt (Thr308) (#2965), and PI3 Kinase p110α (#4249). Anti-GAPDH (mAbcam, #9484) was used as loading control. After overnight incubation, membranes were probed with secondary antibodies. Levels of protein were finally quantified using the Odyssey imaging system from Li-Cor Biosciences (Image Studio Lite Ver 5.2).

Rossello X., Riquelme J.A., He Z., Taferner S., Vanhaesebroeck B., Davidson S.M, & Yellon D.M. (2017). The role of PI3Kα isoform in cardioprotection. Basic Research in Cardiology, 112(6), 66.

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Characterizing Protein Adsorption on PCL Scaffolds

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The protein adsorption on PCL scaffolds was determined by soaking the scaffolds in PBS 1x (pH 7.4) overnight and then in 10% fetal bovine serum (FBS) for 24 hours at 37 °C. The scaffolds were washed three times with PBS 1x for 10 minutes to remove non-specifically adsorbed proteins. Then scaffolds were subjected to a 2% sodium dodecyl sulfate (SDS) solution under shaking conditions (100 rpm, 37 °C) for 6 hours to collect adhered proteins. The supernatant was collected and evaluated using bicinchoninic acid (BCA) protein assay reagent (Sigma Aldrich). The BSA was used as a standard and the supernatant was quantified using an UV-visible spectrophotometer (Varian Cary® 300 Scan; Varian Instruments, CA, USA) at a wavelength of 562 nm. Representative measurements of three distinct sets of data have been reported (Student t- test, P < 0.05).
The recovered serum protein samples were subject to fractionation through 4–12% SDS- polyacrylamide gel electrophoresis (SDS-PAGE). The resolved protein bands were visualized by Coomassie brilliant blue staining (BioRad), according to the manufacturer’s instructions. Representative results of three independent experiments have been reported.

Palamà I.E., Arcadio V., D’Amone S., Biasiucci M., Gigli G, & Cortese B. (2017). Therapeutic PCL scaffold for reparation of resected osteosarcoma defect. Scientific Reports, 7, 12672.

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Cerebral Cortex and Hippocampus Protein Extraction

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Animals were anesthetized with intraperitoneal injection of 2% sodium pentobarbital (40–50 mg/kg), then sacrificed by decapitation. The cerebral cortex and hippocampus were dissected, immediately weighed, frozen in liquid nitrogen, and stored at −80°C. Protein extracts were prepared in RIPA cell lysis buffer (Thermo Fisher Scientific, Inc.). The lysates were sonicated in an iced water bath for 30 min and centrifuged at 37,318.4 × g at 4°C for 20 min to collect the supernatants. The protein concentration was measured using a BCA protein assay reagent (Sigma-Aldrich; Merck KGaA) according to manufacturer's instructions.

Wang Y., Ma Q., Ma X., Zhang Z., Liu N, & Wang M. (2017). Role of mammalian target of rapamycin signaling in autophagy and the neurodegenerative process using a senescence accelerated mouse-prone 8 model. Experimental and Therapeutic Medicine, 14(2), 1051-1057.

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Colonic Protein Expression Analysis

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The end of experiment, Mice were sacrificed and the section of colons (5–8 mm) were dissected and flushed with PBS. Distal colons were homogenized in lysis buffer (iNtRON Biotech, Republic of Korea) and centrifuged at 16,582 g for 5 min. The supernatants were transferred to fresh tubes and protein concentrations were determined using BCA protein assay reagent (Sigma-Aldrich, St. Louis, MO, USA). Lysates (50

μ

g protein) were separated by 10 % SDS-PAGE and transferred to membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA), which were blocked with 5 % skim milk in 0.05 % PBS-Tween-20 (PBST) for 1 h at room temperature. Membranes were incubated overnight with primary antibodies against phospho-extracellular signal regulated kinase (pERK), phospho-3-Jun, N-terminal kinase (p-JNK), p38 (Cell Signaling technology, Danvers, MA, USA) and glyceraldehyde-phosphate dehydrogenase (GAPDH) (Thermo scientific, Waltham, MA, USA) and washed three times with PBST. Blots were incubated with secondary antibody for 1 h at room temperature. Antibody-specific proteins were visualized using an enhanced chemiluminescence detection system (Amersham, Newark, NJ, USA). Protein densities were quantified by densitometry.

Jeon Y.D., Bang K.S., Shin M.K., Lee J.H., Chang Y.N, & Jin J.S. (2016). Regulatory effects of glycyrrhizae radix extract on DSS-induced ulcerative colitis. BMC Complementary and Alternative Medicine, 16, 459.

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BrdU Assay and ALP Activity in BMSCs

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The bromodeoxyuridine (BrdU) assay was conducted to study the cell proliferating effect of the CDR and SrR on UMR106 and BMSCs. The assay was started 24 hours after the drugs had been added.
To measure the alkaline phosphatase (ALP) activity, 2.5 × 10⁵ BMSCs in 1 ml αMEM growth medium were seeded in 6-well plates. The cells were incubated for 6 days. Then, the original medium in the 6-well plate was replaced by osteogenic medium containing 200 μg/ml of CDR or SrR, or 200 μg/ml of CDR with 200 or 400 μg/ml SrR (based on the results of the MTT and BrdU assays). The BMSCs incubated with osteogenic medium without drug acted as control (Ctrl). The cells were cultured continuously and their ALP activity was measured using a commercially available ALP assay kit (Stanbio, USA). Total protein content was determined with BCA protein assay reagent (Sigma, USA) and enzyme activities were expressed as U/mg protein.

Siu W.S., Shiu H.T., Ko C.H., Shum W.T., Yu H.N., Lau C.B., Hung L.K, & Leung P.C. (2017). Integrative Approach to Facilitate Fracture Healing: Topical Chinese Herbal Paste with Oral Strontium Ranelate. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 9795806.

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Quantifying Cytokine Levels by ELISA

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Levels of IL-6 and TNF-α in the serum and tissue were measured using an enzyme-linked immunosorbent assay (ELISA), as previously described [22 (link)]. Briefly, 96-well plates (SPL Life Science, Seoul, Korea) were coated with 100

μ

L of anti-mouse monoclonal antibody (1.0 mg/mL at pH 7.4 in phosphate buffered saline [PBS]) and incubated overnight at 4∘C. After additional washes, 50 μL of sample, or IL-6 and TNF-α (BD Bioscience, San Diego, CA, USA) standard was added and incubated at room temperature for 2 h. Plates were washed and 0.2 μg/mL of biotinylated anti-mouse antibody was added and incubated at room temperature for 2 h. After washing, avidin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA) was added and plates were incubated for 30 min at 37∘C. The plates were then washed again and (2,2′-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) substrate (BD Bioscience, San Diego, CA, USA) was added. Color development was measured at 405 nm using an automated microplate ELISA reader (Molecular Devices, Sunnyvale, CA, USA). Standard curves were prepared using serial dilutions of recombinant antibody (BD Bioscience, San Diego, CA, USA). Protein concentrations were measured using bicinchoninic acid (BCA) protein assay reagent (Sigma-Aldrich, St. Louis, MO, USA).

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Extraction of Salt-soluble Beef ER Proteins

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Salt-soluble proteins were extracted from beef ER according to the modified method of DeFreitas et al. (1997) (link). Briefly, one part of thawed beef ER samples and three parts of isolation buffer (0.5 M NaCl, 17.8 mM Na₅P₃O₁₀, pH 8.3) were homogenized in a blender (SMT PH 91, SMT, Japan) for 60 s at medium speed. The slurry was kept at 4°C for 2 h, followed by centrifugation (12,000 rpm) at 4°C for 60 min (1736R, Labogene, Korea). The extract was then filtered through several layers of cheesecloth. Protein concentrations of filtered supernatant were determined using the BCA Protein Assay reagent (Sigma-Aldrich, USA).

Kim H., Park D.H., Hong G.P., Lee S.Y., Choi M.J, & Cho Y. (2018). The Change in Quality Characteristics of Hanwoo in Home Meal Replacement Products under Different Cooking and Freezing Methods. Korean Journal for Food Science of Animal Resources, 38(1), 180-188.

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Proteomic Profiling of Cellular Biomolecules

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Solutions were prepared with Milli-Q water. Water of HPLC grade, trifluoroacetic acid (TFA), isopropyl alcohol, tributyl phosphate (TBP), ammonium bicarbonate (ABC), dithiothreitol (DTT), iodoacetamide (IAA), urea, Triton X-100, Tween-20, Trizma-base, MES, sucrose, BCA protein assay reagent and BSA were from SIGMA Chemical Co. (St. Louis, MO, USA). Acetonitrile (ACN), methanol, and glycerol were from Merck (Darmstadt, Germany). Porcine trypsin was from Promega (Madison, WI, USA). Bradford proteinbinding colorimetric assay was purchased from Bio-Rad (Hercules, CA, USA). Anti-protease inhibitor cocktail (Complete) was from Roche (Monza, Italy). NuPAGE s SDS-PAGE gel electrophoresis system components (mini gels, running and loading buffers, molecular weight markers and Coomassie brilliant blue staining-CBB) were supplied by Life Technologies (Paisley, Renfrewshire, UK). Hybond-ECL nitrocellulose membrane was from GE (Little Chalfont, Buckinghamshire, UK). Anti-aminopeptidase N (CD13) monoclonal antibody, anti-annexin A2 (ANXA2) and anti-carbonic anhydrase (CA2) polyclonal antibodies were from Abcam (Cambridge, UK). Species-specific secondary peroxidase conjugated antibodies and ECL reagents were from Pierce (Rockford, IL, USA).

Raimondo F., Corbetta S., Savoia A., Chinello C., Cazzaniga M., Rocco F., Bosari S., Grasso M., Bovo G., Magni F, & Pitto M. (2015). Comparative membrane proteomics: a technical advancement in the search of renal cell carcinoma biomarkers. Molecular bioSystems, 11(6).

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Mitochondrial Protein Isolation and Analysis

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ARPE cells were scratched and were collected for the protein isolation of mitochondrial and cytosol fractions with Mitochondria/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA), according to the kit's manual. Protein concentration was titered using the BCA protein assay reagent (Sigma-Aldrich). Protein samples were separated with 12% SDS-PAGE gel, and were transferred to a PVDF Transfer Membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was inoculated with a rabbit polyclonal antibody to cytochrome c, caspase 3, caspase 9, Cox4l1 or β-actin (either with 1:1000, all from Sigma-Aldrich) for the antigen-antibody binding. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL, USA)and enhanced chemil u m i n e s c e n c e ( E C L ) ( A m e r s h a m P h a r m a c i a Biotech, Amersham, UK) were utilized to visualize the Ag-Ab binding.

Yan G., Yu L., Jiang S, & Zhu J. (2018). Melatonin antagonizes oxidative stress-induced mitochondrial dysfunction in retinal pigmented epithelium cells via melatonin receptor 1 (MT1). The Journal of toxicological sciences, 43(11).

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Bacteriocin Purification from Bioreactor CFS

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The pH of a 100-mL aliquot of CFS from the bioreactor was adjusted to 6.0 with 1.0 M NaOH and was subsequently used for further assays. Bacteriocin ST16Pa was precipitated by adding ammonium sulfate to CFS in order to obtain 30% (wt/vol) saturation and then stirred at 4°C for 2 h. The pellet resulting from centrifugation at 4,470 × g at 4°C for 30 min was resuspended in 10 mL of 25 mM ammonium acetate buffer (pH 6.5) and loaded on a previously activated C 18 solid phase extraction (SPE) cartridge (model Oasis HLB; Waters, Millipore), which was eluted with isopropanol solutions in the same buffer with gradually increasing concentration [20, 40, 60, and 80% (vol/vol) ]. To control and validate the proposed protocol, Lb. plantarum ST16Pa was cultured in MRS broth at 30°C for 24 h, and the resulting CFS was treated as previously described.
The antimicrobial activity was tested against L. innocua 6a CLIST 2865 by the spot-on-the-lawn method as previously described. The protein concentration after each step was determined using the BCA protein assay reagent (Sigma-Aldrich) as specified by the manufacturer and a calibration curve (y = 0.0011 x + 0.00245; R 2 = 0.99). It was possible to determine the total antimicrobial activity (AU), specific activity (AU/mL), yield (%), and purification fold.

Sabo S.S., Converti A., Ichiwaki S, & Oliveira R.P.S. (2019). Bacteriocin production by Lactobacillus plantarum ST16Pa in supplemented whey powder formulations. Journal of dairy science, 102(1).

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