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Bca protein concentration kit

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The BCA protein concentration kit is a colorimetric assay used to quantify the total protein concentration in a sample. It uses bicinchoninic acid (BCA) to detect the presence of protein. The kit provides a simple, reliable, and reproducible method for measuring protein levels in a variety of sample types.

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Lab products found in correlation

Trypsin, by Thermo Fisher Scientific (4 mentions) Lys c protease, by Fujifilm (4 mentions) Protease inhibitor tablet, by Thermo Fisher Scientific (3 mentions) Tandem mass tag isobaric reagents, by Thermo Fisher Scientific (2 mentions) Sep pak cartridges 50 mg, by Waters Corporation (2 mentions) By4716, by Thermo Fisher Scientific (2 mentions) Nrrl y 164, by American Type Culture Collection (2 mentions) Stagetip empore c18 material, by 3M (2 mentions) Chloroform, by Maclin Biochemical Technology (1 mentions) Dmem medium, by Thermo Fisher Scientific (1 mentions) Dpbs solution, by Thermo Fisher Scientific (1 mentions) Jc 1 kit, by Beyotime (1 mentions) Gallic acid, by Yuanye Bio-Technology (1 mentions) Alkaline phosphatase staining kit, by Beyotime (1 mentions) Exosome free serum, by System Biosciences (1 mentions) Sep pak cartridges, by Waters Corporation (1 mentions) Pierce c18 tip, by Thermo Fisher Scientific (1 mentions) Tandem mass tag tmtpro isobaric reagents, by Thermo Fisher Scientific (1 mentions) Luria bertani broth lb, by Merck Group (1 mentions) High select fe nta phosphopeptide enrichment kit, by Thermo Fisher Scientific (1 mentions)

9 protocols using bca protein concentration kit

1

Quantifying KIF20A Protein Expression in U251 Cells

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Total protein was extracted from U251 cells and quantified using the BCA protein concentration kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, equal amounts of proteins were separated by electrophoresis on SDS-PAGE gels and transferred onto nitrocellulose membranes. Immunoblotting was performed using the following antibodies: Rabbit anti-KIF20A (ab70791, 1:2,000 dilution) and anti-GAPDH (ab8245, 1:500 dilution) at 4 °C overnight. Next, the membranes were washed repeatedly and then incubated with secondary antibody. The Western blot bands were visualized and analyzed by ImageJ following standard methods.
Fan P., Xia J., Zhou M., Zhuo C, & He H. (2023). Development and validation of a personalized classifier to predict the prognosis and response to immunotherapy in glioma based on glycolysis and the tumor microenvironment. PeerJ, 11, e16066.
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2

Exosomal Regulation of Osteoblast Function

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Gallic acid (Yuanye BioTech, Shanghai, China); Type I collagenase, CAT ELISA kit, SIRT1 ELISA kit (Sigma, St. Louis, MO, USA); Exosome-free serum (SBI, Palo Alto, CA, USA); Medium DMEM, DPBS solution (Gibco, Grand Island, NY, USA); chloroform, isopropanol (MackLin, Shanghai, China); SOD1, SOD2 ELISA kit (Ray Biotech, Guangzhou, Guangdong, China); SIRT3 ELISA kit (Abnova, Taipei, Taiwan, China); MTT, ATP kit, alizarin red dye solution (Solarbio, Beijing, China); lactic acid content kit (Ruixin Biotech, Quanzhou, Fujian, China); JC-1 kit (Beyotime, Shanghai, China); carbonyl cyanide m-chlorophenylhydrazone CCCP (BestBio, Shanghai, China); MDA kit (Dojindo, Kumamoto, Japan); SOD kit (Jingmei Biological, Yancheng, Jiangsu, China); BCA protein concentration kit (Thermo, Waltham, MA, USA); alkaline phosphatase staining kit (Beyotime, Shanghai, China); anti-CD81, Anti-CD9, Anti-ALIX, Anti-β-actin (Abcam, Cambridge, MA, USA). Primers were ordered from Shanghai Sangon Biotech.
Dai Z., Li Z., Zheng W., Yan Z., Zhang L., Yang J., Xiao J., Sun H., Li S, & Huang W. (2022). Gallic Acid Ameliorates the Inflammatory State of Periodontal Ligament Stem Cells and Promotes Pro-Osteodifferentiation Capabilities of Inflammatory Stem Cell-Derived Exosomes. Life, 12(9), 1392.
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3

Automated TMT Proteomics Workflow

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Tandem Mass Tag (TMTpro) isobaric reagents, BCA protein concentration kit, protease inhibitor tablets, Pierce C18 tips, and trypsin were purchased from ThermoFisher Scientific (Rockford, IL). StageTip Empore-C18 material was purchased from CDSanalytical (Oxford, PA). Sep-Pak cartridges (100 mg) were from Waters (Milford, MA). Lys-C protease was from Fujifilm Wako (Richmond, VA). Mass spectrometry grade water and organic solvents were from J.T. Baker (Center Valley, PA). The S. cerevisiae strains were from Horizon Scientific (Cambridge, UK). Synthetic complete (SC) media was from Sunrise Science (San Diego, CA). Sera-Mag Speed Beads (cat. nos. 45152105050350 and 65152105050350) were from GE Life Sciences (Marlborough, MA).
The OT-2 robotic liquid handler was purchased from opentrons (New York, NY) and included the magnetic module, the temperature module, and two GEN2 8-channel pipets: 20 and 300 μL. Labware used on the OT-2 included: 12-channel reservoirs (USA Scientific, 1061-8150), 96-well microplate (Bio-Rad, HSP9601), 2 mL deep 96-well microplates (USA Scientific, 1896-2000), and 8-tube strip PCR tubes (Bio-Rad, TLS0801). Also, 8-tube strip PCR tube caps (Bio-Rad, TLS0803) are required for 37 °C incubations. Proteolytic digests were performed on the Jitterbug Heated Microplate Shaker (Boekel Scientific, 130000).
Liu X., Gygi S.P, & Paulo J.A. (2021). A Semiautomated Paramagnetic Bead-Based Platform for Isobaric Tag Sample Preparation. Journal of the American Society for Mass Spectrometry, 32(6), 1519-1529.
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4

Quantitative Proteomics of Microbes

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Tandem Mass Tag (TMT) isobaric reagents, BCA Protein Concentration Kit, Protease inhibitor tablets, and trypsin were purchased from ThermoFisher Scientific (Rockford, IL). StageTip Empore-C18 material was purchased from CDS (Oxford, PA). Sep-Pak cartridges (50 mg) were from Waters (Milford, MA). LysC protease was from Fujifilm Wako (Richmond, VA). Mass spectrometry-grade water and organic solvents were from J.T. Baker (Center Valley, PA). The S. cerevisiae strain used was BY4716 from ThermoFisher Scientific (Waltham, MA). The S. pombe strain was NRRL Y-164 purchased from ATCC (Manassas, Virginia). The E. coli strain was purchased from Carolina Biological Supply Co. (Burlington, NC). Synthetic complete (SC), Edinburgh Minimal Media (EMM), yeast extracts with supplements (YES), and yeast-peptone-dextrose (YPD) media was from Sunrise Science (San Diego, CA). Luria-Bertani broth (LB) and Nutrient broth #1 (NB) was purchased from Millipore-Sigma (St. Louis, MO). Media formulations are available in the Supplemental Methods. Sera-MagSpeed Beads (cat. nos. 45152105050350 and 65152105050350) were from GE Life Sciences (Marlborough, MA).
Navarrete-Perea J., Gygi S.P, & Paulo J.A. (2020). Growth media selection alters the proteome profiles of three model microorganisms. Journal of proteomics, 231, 104006.
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5

Tandem Mass Tag Proteomics

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Tandem Mass Tag (TMT) isobaric reagents, BCA Protein Concentration Kit, Protease inhibitor tablets, and trypsin were purchased from ThermoFisher Scientific (Rockford, IL). StageTip Empore-C18 material was purchased from 3M (St. Paul, MN). Sep-Pak cartridges (50 mg) were from Waters (Milford, MA). Lys-C protease was from Fujifilm Wako (Richmond, VA). Mass spectrometry-grade water and organic solvents were from J.T. Baker (Center Valley, PA). The S. cerevisiae strain used was BY4716 from ThermoFisher Scientific (Waltham, MA). The S. pombe strain was NRRL Y-164 purchased from ATCC (Manassas, Virginia). Synthetic complete (SC) and Edinburgh Minimal Media (EMM) media was from Sunrise Science (San Diego, CA). Glucose and pyruvate were from MilliporeSigma (St. Louis, MO).
Paulo J.A., Navarrete-Perea J, & Gygi S.P. (2019). Multiplexed Proteome Profiling of Carbon Source Perturbations in Two Yeast Species with SL-SP3-TMT. Journal of proteomics, 210, 103531.
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6

Phosphoproteomics Sample Preparation

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TMT reagents, BCA protein concentration kit, trypsin and High-Select Fe- NTA phosphopeptide enrichment kit were from ThermoFisher Scientific (Rockford, IL). StageTip Empore C-18 material was from 3 M (Saint Paul, MN). SepPak cartridges (100 mg) were from Waters (Mildford, MA). Lys-C protease was from Wako (Boston, MA). Water and organic solvents were from J.T. Baker (Center Valley, PA). cOmplete protease and PhosStop phosphatase inhibitors were from Millipore-Sigma (Saint Louis, MO). Yeast synthetic complete media was from Sunrise Science (San Diego, CA), bathopenanthrolinedisulfonic acid (BPS) was from Sigma (St. Louis, MO). Unless otherwise noted, all the other chemicals were from ThermoFisher Scientific (Waltham, MA).
Navarrete-Perea J., Guerra-Moreno A., Van Vranken J., Isasa M., Paulo J.A, & Gygi S.P. (2021). Iron Deficiency and Recovery in Yeast: A Quantitative Proteomics Approach. Journal of proteome research, 20(5), 2751-2761.
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7

Tetrazolium-Based SOD Activity Assay

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The SOD activity was detected through a microtiter plate assay method with some modifications using a water-soluble tetrazolium salt (WST-1) (18 (link)). Sample solution of 20 μL was added into 200 μL 3 mM water-soluble tetrazolium solution and 20 μL 29 mU xanthine oxidase solution. After incubation at 37°C for 20 min, the absorbance at 450 nm was detected.
Protein content was assayed with a BCA protein concentration kit (Thermo Scientific, USA), using bovine serum protein as a calibration curve standard.
Zhao Y., Zhao L., Zhang W., Rao L., Wang Y, & Liao X. (2022). Production of a Novel Superoxide Dismutase by Escherichia coli and Pichia pastoris and Analysis of the Thermal Stability of the Enzyme. Frontiers in Nutrition, 9, 850824.
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8

Quantifying ATP Levels in C2C12 Cells

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C2C12 mouse myoblasts in 6-well plates were fully lysed with a lysis buffer, and the supernatant was collected by centrifugation (12000 g, 4°C, 5 min). ATP content in each sample was examined using an Enhanced ATP Assay Kit (Beyotime, Beijing, China) by a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) and normalized by protein concentration which was quantified by a BCA protein concentration kit (Thermo Fisher Scientific, Waltham, MA, USA).
Dong W., Chen W., Zou H., Shen Z., Yu D., Chen W., Jiang H., Yan X, & Yu Z. (2022). Ginsenoside Rb1 Prevents Oxidative Stress-Induced Apoptosis and Mitochondrial Dysfunction in Muscle Stem Cells via NF-κB Pathway. Oxidative Medicine and Cellular Longevity, 2022, 9159101.
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9

Mitochondrial ROS Measurement by MitoSOX

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Mitochondrial ROS production was measured by MitoSOX Red fluorescence. Inducible TOLLIP overexpressing cells and control cells were setup in 6 well plate at a 2 × 105/well, treated by doxycycline at 2 μg/ml for 48 hours. For the knocking down experiment, BEAS-2B cells were seeded in 6 well plate at a 4 × 105/well, transfected with TOLLIP siRNA and scramble siRNA for 48 hours. Then all cells were treated with either PBS (control) or bleomycin at 50 μM (Sigma B8416) for 8 hours. After treatment, cells were collected and resuspended in 20 μl culture media. Cell suspension were transferred into each well (10 μl/well) of a 96-well plate and incubated with MitoSOX (10 nM, Invitrogen,) for 10 minutes. The cells were washed with PBS and fluorescent intensity (510/580 nm) was measured kinetically. The fluorescent intensity of each well was normalized by total protein measured using BCA protein concentration kit (Thermo Scientific).
Li X., Kim S.E., Chen T.Y., Wang J., Yang X., Tabib T., Tan J., Guo B., Fung S., Zhao J., Sembrat J., Rojas M., Shiva S., Lafyatis R., Croix C.S., Alder J.K., Di Y.P., Kass D.J, & Zhang Y. (2020). Toll Interacting Protein Protects Bronchial Epithelial Cells from Bleomycin-Induced Apoptosis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(8), 9884-9898.
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