Bicinchoninic protein assay, by Pierce - Product detail - Pubcompare (2024)

Manufactured by Pierce

Sourced in United States

The Bicinchoninic Protein Assay is a colorimetric detection and quantitation method used to determine the total protein concentration in a sample. It is based on the reduction of copper ions by proteins in an alkaline medium and the subsequent chelation of the reduced copper ions with bicinchoninic acid (BCA) to produce a purple-colored complex that absorbs light at 562 nm. This assay provides a simple, sensitive, and stable method for the quantitation of protein levels.

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Lab products found in correlation

IBright Analysis Software, Invitrogen (2 mentions) Radiance Plus, Bio-Rad (2 mentions) VersaDoc MP5000, Bio-Rad (2 mentions) IBright 1500, Invitrogen (2 mentions) TissueLyser II, Qiagen (2 mentions) Quantity One software, Bio-Rad (2 mentions) RIPA buffer, Abcam (1 mentions) Amersham ECL Prime Western Blotting Detection Reagent, General Electric (1 mentions) Amicon Ultra-15 filter, Millipore (1 mentions) Enhanced chemiluminescence western blotting detection system, General Electric (1 mentions) RIPA, Thermo Fisher (1 mentions) Albumin diagnostic kit, Wiener Lab (1 mentions) Wiener reagents and procedures, Wiener Lab (1 mentions)

8 protocols using Bicinchoninic protein assay

Spleen and kidney tissues were homogenized using a RIPA buffer (Abcam, catalog#156034) and protein concentrations determined using a bicinchoninic protein assay (Pierce). SDS-PAGE electrophoresis was conducted with 4–20% gradient polyacrylamide gels. Membranes were exposed to antibodies targeted against GAPDH, Very-Late Antigen -4 (VLA-4), Vascular Cell Adhesion Molecule-1 (VCAM-1), LLT1, Monocyte Chemoattractant Protein-1 (MCP-1), Intercellular Adhesion Molecule-1 (ICAM-1), α7nAChR, Angiotensin II receptor-Type 1 (AT1R), and Angiotensin II receptor-Type 2 (AT2R). Signal detection was accomplished using AmershamECLPrime Western Blotting Detection Reagent (GE Lifesciences, product # RPN2236).

Harwani S.C., Ratcliff J., Sutterwala F.S., Ballas Z.K., Meyerholz D.K., Chapleau M.W, & Abboud F.M. (2016). Nicotine Mediates CD161a+ Renal Macrophage Infiltration and Premature Hypertension in the Spontaneously Hypertensive Rat. Circulation research, 119(10), 1101-1115.

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E. coli BL21 (DE3) was transformed with pET28-luxS and grown in LB medium supplemented with 50 μg/mL kanamycin at 37°C until an optical density at 600 nm of 0.8 was reached. Expression of LuxS was induced by the addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 5 h in the culture at 37°C. The cells were harvested by centrifugation and lysed by sonication (on ice) in 20 mM Tris-HCl (pH 8.0), 0.2M NaCl, 10 mM imidazole, 0.1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. The crude lysate was clarified by two centrifugations at 60,000 g for 30 min at 4°C and filtration through a 0.45- mm membrane. Soluble protein was purified by HisTrap column (GE) followed by size-exclusion chromatography on a Superdex 200 FPLC column (Amersham Biosciences). Fractions containing LuxS were pooled and concentrated in an Amicon Ultra-15 filter (Millipore) using a 10-kDa cutoff membrane. Protein purity was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentrations were determined using a bicinchoninic protein assay according to the manufacturer’s instructions (Pierce).

Wang Y., Yi L., Wang S., Fan H., Ding C., Mao X, & Lu C. (2015). Crystal Structure and Identification of Two Key Amino Acids Involved in AI-2 Production and Biofilm Formation in Streptococcus suis LuxS. PLoS ONE, 10(10), e0138826.

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After harvesting of the cells, protein extracts were prepared using lysis buffer containing RIPA buffer, with protease and phosphatase inhibitors. Protein concentration in the cell lysate was measured by the bicinchoninic protein assay (Pierce Biotechnology Inc., Rockford, IL, USA). An equal amount of protein for all samples was subjected to electrophoresis and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in PBS containing 0.1% Tween 20 for 20min and incubated at 4°C overnight with primary antibodies for HO-1, IRG1, A20 and β-actin, followed by secondary antibodies conjugated with horseradish peroxidase. Immunocomplexes were visualized using the enhanced chemiluminescence western blotting detection system (GE Healthcare Life Sciences, Buckinghamshire, UK).

Jamal Uddin M., Joe Y., Kim S.K., Oh Jeong S., Ryter S.W., Pae H.O, & Chung H.T. (2015). IRG1 induced by heme oxygenase-1/carbon monoxide inhibits LPS-mediated sepsis and pro-inflammatory cytokine production. Cellular and Molecular Immunology, 13(2), 170-179.

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Frozen heart tissues were homogenized with a bead mill (Qiagen, TissueLyserII) in tissue lysis buffer (Table

S1

). Homogenates were sonicated, clarified by centrifugation (16000g, 10minutes, 4 °C), and normalized by concentration using the bicinchoninic protein assay (Pierce, 23225). Equal masses of protein were separated, transferred, and analyzed using standard western blot techniques with antibodies (Table

S1

). The loading ranged from 20 to 50µg of total protein per lane, depending on several variables (gel type, size, well format, sample amount, and antibody quality/sensitivity). Most western blots used Biorad Stain‐Free gels (item 5678115) with total protein imaged on the gel after running, and membrane after transfer to quantify equal loading and transfer. Others used Nupage 4% to 12% Bis‐Tris gels (item NP0336). Images were acquired using enhanced chemiluminescence substrate (Azure, Radiance Plus) on a Biorad Versadoc MP5000 and quantified using Biorad QuantityOne Software (Version 4.6.6) with global background subtraction settings or acquired using chemiluminescence and/or dual‐channel fluorescence on an Invitrogen iBright‐1500 and quantified using iBright Analysis Software (Version 4.0.0).

McLendon J.M., Zhang X., Matasic D.S., Kumar M., Koval O.M., Grumbach I.M., Sadayappan S., London B, & Boudreau R.L. (2022). Knockout of Sorbin And SH3 Domain Containing 2 (Sorbs2) in Cardiomyocytes Leads to Dilated Cardiomyopathy in Mice. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 11(13), e025687.

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MM cell lines MM1S or DOX40 were incubated with LBH589 or RAD001 or the combination for the indicated time points and lysed using RIPA buffer (50mM HEPES (pH 7.4), 150mM NaCl, 1% Triton X-100, 30mM sodium pyrophosphate, 5mM EDTA, 2mM Na3VO4, 5mM NaF, 1mM phenylmethyl-sulfonyl-fluoride (PMSF) and protease inhibitor cocktail). Protein concentrations in the lysate were measured by bicinchoninic protein assay (Pierce, Rockford, IL, USA). Equal amounts of protein were loaded onto a SDS-PAGE gel and transferred onto a nitrocellulose membrane. Membranes were probed with individual antibodies and antigen-antibody complexes were detected using enhanced chemiluminescence (Amersham, Arlington Heights, IL). All antibodies except those mentioned below were purchased from Cell Signaling Technologies (Daverns, MA). Puma antibody was purchased from ProSci Inc. (Poway, CA) and AcH3 and AcH4 antibodies were purchased from Millipore (Billerica, MA).

Ramakrishnan V., Kimlinger T., Timm M., Haug J., Rajkumar S.V, & Kumar S. (2014). Multiple mechanisms contribute to the synergistic anti-myeloma activity of the pan-histone deacetylase inhibitor LBH589 and the rapalog RAD001. Leukemia research, 38(11), 1358-1366.

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For synchronization in anaphase, cells were arrested by treatment with STLC for 16 hr followed by addition of Mps1i for the times indicated in the figure legends. Cells were harvested and washed once in PBS. Cell pellets were resuspended in radioimmunoprecipitation buffer (RIPA, ThermoFisher) supplemented with 1X Complete EDTA-free protease inhibitor cocktail (Roche), 1 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerophosphate, 1 mM sodium fluoride, and 0.4 mM sodium orthovanadate. Cells were lysed on ice for 15 min, and cellular debris was removed by centrifugation at >10,000 g for 10 min at 4°C. Protein concentrations were measured and normalized using a bicinchoninic protein assay (Pierce). Samples were analyzed by SDS-PAGE and Western blot.

Maier N.K., Ma J., Lampson M.A, & Cheeseman I.M. (2021). Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition. Developmental cell, 56(15), 2192-2206.e8.

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Protein content was measured by the bicinchoninic protein assay (Pierce Biotechnology Inc., Rockford, IL, USA). Albumin content was determined colorimetrically based on albumin binding to bromocresol green using an albumin diagnostic kit (Wiener Lab, Buenos Aires, Argentina).
LDH activity, expressed as units per liter of BAL fluid, was determined by measuring the formation of the reduced form of nicotinamide adenine dinucleotide using the Wiener reagents and procedures (Wiener Lab). The lung wet:dry weight ratio was measured as previously obtained and described by Aeffner et al. [18] . Briefly, mice were euthanized and exsanguinated, and their lungs removed, weighed, and dried in an oven at 55 °C for 7 days. After drying, the lungs were weighed again. Wet:dry weight ratio was then calculated as an index of intrapulmonary fluid accumulation, without correction for blood content.
Whole-lung samples from all experimental groups were excised and washed out with PBS. Then, tissues were immersed in 4 % (v/v) formalin saline solution. Once fixed, samples were dehydrated and embedded in Histowax (Leica Microsystems Nussloch GmbH, Nussloch, Germany) at 56 °C. Finally, lungs were cut into 4 lm serial sections and stained with hematoxylin-eosin for light microscopy examination. All slides were coded and evaluated blindly.

Zelaya H., Tada A., Vizoso-Pinto M.G., Salva S., Kanmani P., Agüero G., Alvarez S., Kitazawa H, & Villena J. (2015). Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage. Inflammation research : official journal of the European Histamine Research Society ... [et al.], 64(8).

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Frozen heart tissues were homogenized with a bead mill (Qiagen, TissueLyserII) in tissue lysis buffer (Supp. Table 1). Homogenates were sonicated, clarified by centrifugation (16,000×g, 10 mins, 4℃), and normalized by concentration using the bicinchoninic protein assay (Pierce, 23225). Equal masses of protein were separated, transferred, and analyzed using standard Western Blot techniques with antibodies (Supp. Table 1). The loading ranged from 20 to 50 ug of total protein per lane depending on several variables (gel type, size, well format, sample amount, Ab quality/sensitivity, etc.). Most western blots used Biorad Stain-Free gels (item 5678115) with total protein imaged on the gel after running, and membrane after transfer to quantify equal loading and transfer. Others used Nupage 4-12% Bis-Tris gels (items NP0336).
Images were acquired using enhanced chemiluminescence substrate (Azure, Radiance Plus) on a Biorad Versadoc MP5000 and quantified using Biorad QuantityOne Software (Version 4. 6.6) with global background subtraction settings or acquired using chemiluminescence and/or Dualchannel fluorescence on an Invitrogen iBright-1500 and quantified using iBright Analysis Software (Version 4.0.0).

McLendon J.M., Zhang X., Matasic D.S., Kumar M., Koval O.M., Grumbach I.M., Sadayappan S., London B., & Boudreau R.L. (2022). Knockout of Sorbs2 in Cardiomyocytes Leads to Dilated Cardiomyopathy in Mice.

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Bicinchoninic protein assay, by Pierce - Product detail - Pubcompare (2024)
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