Pierce bca assay

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany, Canada, Switzerland

The Pierce BCA assay is a colorimetric detection method for quantifying total protein concentration in a sample. It relies on the reduction of Cu2+ to Cu+ by protein in an alkaline medium, with the subsequent chelation of the Cu+ ions by bicinchoninic acid (BCA) to produce a purple-colored product that absorbs light at 562 nm.

Automatically generated - may contain errors

Lab products found in correlation

Ripa buffer, by Thermo Fisher Scientific (22 mentions) Nitrocellulose membrane, by Bio-Rad (10 mentions) β actin, by Cell Signaling Technology (9 mentions) Protease inhibitor cocktail, by Merck Group (9 mentions) Odyssey infrared imaging system, by LI COR (8 mentions) Ripa buffer, by Merck Group (8 mentions) β actin, by Merck Group (7 mentions) Gapdh, by Cell Signaling Technology (6 mentions) Chemidoc mp imaging system, by Bio-Rad (6 mentions) Pvdf membrane, by Bio-Rad (6 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (6 mentions) Pvdf membrane, by Merck Group (6 mentions) Pierce ecl, by Thermo Fisher Scientific (6 mentions) Turbo blot transfer system, by Bio-Rad (5 mentions) Image lab software, by Bio-Rad (5 mentions) β actin, by Santa Cruz Biotechnology (5 mentions) Trans blot turbo rta transfer kit, by Bio-Rad (4 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions) Protease inhibitor cocktail, by Roche (4 mentions) Nitrocellulose membrane, by GE Healthcare (4 mentions)

318 protocols using pierce bca assay

Brain Tissue Homogenization and Protein Extraction

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Frozen tissue (400–600 mg) was homogenized in 1 ml ice-cold brain lysis buffer (BLB: 10 mM Tris, 1 mM EDTA, 0.8 M NaCl, 10% Sucrose, pH 7.4, EDTA-free protease inhibitors) using a Precellys homogeniser (5,000 rpm, 2 × 20 s, break: 15 s). Then, the buffer volume was adjusted to 10-fold of volume, Benzonase (0.25 U/μl) was added and the homogenate incubated for 1 h at 4°C. Protein concentration was determined with the Pierce BCA assay (Thermo Fisher Scientific, Waltham, MA, United States) using BSA as a standard. Homogenates were aliquoted and stored at −80°C until further use. For FRASE assays, samples in BLB buffer were used.
For preparation of mouse brain homogenates in RIPA buffer, 400–600 mg brain was homogenized in 1 ml ice-cold 50 mM Tris pH 7.5 buffer using the Precellys homogenizer (5000 rpm, 2×20 s, break: 15 s). Then, buffer volume was adjusted to 10-fold of volume using 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton, 0.1% SDS, 0.5% Sodium deoxycholate, EDTA-free protease inhibitors and Benzonase (0.25 U/μl). Homogenates were incubated for 1 h at 4°C. Protein concentration was determined using the Pierce BCA assay (Thermo Fisher Scientific, Waltham, MA, United States). Homogenates were aliquoted and stored at −80°C until further use.

Schindler F., Praedel N., Neuendorf N., Kunz S., Schnoegl S., Mason M.A., Taxy B.A., Bates G.P., Khoshnan A., Priller J., Grimm J., Maier M., Boeddrich A, & Wanker E.E. (2021). Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington’s Disease Knock-in Mice. Frontiers in Neuroscience, 15, 682172.

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Multiplex Immunoassay Protocol for Protein Analysis

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The following drugs and chemicals were obtained from the sources indicated: CORT (Steraloids Inc., Newport, RI, USA), CPO (Chem Service, Inc., West Chester, PA, USA), DFP (Sigma-Aldrich Co. St. Louis, MO, USA), ethanol (Sigma-Aldrich), phenylmethylsulfonyl fluoride (Sigma-Aldrich), and cell lysis buffer (Bio-Rad, Hercules, CA, USA). A Pierce BCA assay was obtained from Fisher Scientific (Pittsburgh, PA, USA) and includes albumin standards and working reagent. A custom Bio-Plex Pro multiplexed magnetic bead-based immunoassay kit was purchased from Bio-Rad. The custom kit included capture antibodies, biotinylated secondary antibody, standard diluents, streptavidin-phycoerythrin, resuspension buffer, wash buffer, cell lysis buffer, and positive and negative controls.

Penatzer J.A., Miller J.V., Prince N., Shaw M., Lynch C., Newman M., Hobbs G.R, & Boyd J.W. (2021). Differential phosphoprotein signaling in the cortex in mouse models of Gulf War Illness using corticosterone and acetylcholinesterase inhibitors. Heliyon, 7(7), e07552.

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Protein Expression and Pulldown Assay

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Equal amounts of protein,
as determined by Pierce BCA assay (Fisher Scientific), were separated
on 4–20% Tris-glycine gels (BioRad) and transferred to nitrocellulose
membranes.
Expression of the sfGFP reporter and aminoacyl-tRNA
synthetases was confirmed by immunoblotting with antibodies against
GFP (Santa Cruz, sc-9996), FLAG-HRP (Sigma, A8592), β-actin
(cell signaling), and corresponding secondary HRP-conjugated antibodies
when needed (BioRad). For GFP pulldown, GFP-Trap_M magnetic beads
(ChromoTek) were used. For tetrazine pulldown, tetrazine-agarose was
used (CLK-1199-2, Jena Bioscience).

Meineke B., Heimgärtner J., Lafranchi L, & Elsässer S.J. (2018). Methanomethylophilus alvus Mx1201 Provides Basis for Mutual Orthogonal Pyrrolysyl tRNA/Aminoacyl-tRNA Synthetase Pairs in Mammalian Cells. ACS Chemical Biology, 13(11), 3087-3096.

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Protein Extraction and Western Blot Analysis

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Total protein from cultured neutrophils was extracted using the RIPA buffer (BP-115, Boston BioProdcuts, Ashland, MA) with protease inhibitor and phosphatase inhibitor. Protein concentrations were determined using Pierce BCA assay (PI23223, Fisher Scientific). Equal amount of proteins (20 μg per lane) was separated on a 4-20% Mini-PROTEAN TGX Gel (456-1096, Bio-Rad Laboratories, Hercules, CA). Separated proteins were transferred to PVDF membranes using the Transfer Turbo Blot system (Bio-Rad Laboratories) and the Trans-Blot Turbo RTA transfer kit (170-4272, Bio-Rad Laboratories). Membranes were blocked with 5% nonfat milk for 1 hr at room temperature. After blocking, the membranes were incubated overnight at 4 °C with antibodies against IL6 (1:1000, 12912), phosphor-c-Jun N-terminal kinase (p-JNK, 1:1000, 9255), total (t)-JNK (1:1000, 9252,), p-p38 (1:1000, 9211), t-p38 (1:1000, 9212), phosphor-extracellular-signal-regulated kinase (p-ERK1/2, 1:1000, 9101), t-ERK1/2 (1:1000, 9107), and β-actin (1:4000, 4970), all from Cell Signaling Technology. Proteins on the membranes were visualized by chemiluminescence detection kit (Super signal west femto maximum sensitivity substrate, Fisher Scientific). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze signal abundance.

Li J., Deng Z., Zhang X., Liu F., Yang C, & Shi G.P. (2020). Deficiency of IgE protects mice from experimental abdominal aortic aneurysms. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(2), 3091-3104.

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Western Blotting for Protein Analysis

Cited 1 time

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Western blotting was conducted as recently published (Neuhaus et al., 2014 (link)). In brief, cells were scraped after the treatments in RIPA buffer on ice. In case of membran protein enrichment, proteins were extracted with 1% Triton-X 100 followed by lysis of the residual proteins in RIPA buffer. All lysis buffers were supplemented with protease inhibitor cocktail and PhosphoSTOP. Protein concentrations were measured by a detergent-compatible Pierce BCA assay (Fisher Scientific). Twenty microgram protein of total lysates or 10 μg of Triton-X 100 and RIPA fractions per lane were loaded onto 7.5, 10, or 12% SDS–PAGE gels. After gel electrophoresis proteins were immunoblotted onto polyvinylidene difluoride membranes. Incubations with primary and secondary antibodies (Supplementary Table S2) were carried out as previously described (Neuhaus et al., 2012b (link)). ECL-solutions were used for the visualization of the bands using a FluorChem FC2 Multiimager II (Alpha Innotech, Hessisch Oldendorf, Germany). Density values of single protein bands were calculated with the software Alpha View and were related to the corresponding β-actin bands.

Neuhaus W., Krämer T., Neuhoff A., Gölz C., Thal S.C, & Förster C.Y. (2017). Multifaceted Mechanisms of WY-14643 to Stabilize the Blood-Brain Barrier in a Model of Traumatic Brain Injury. Frontiers in Molecular Neuroscience, 10, 149.

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Quantifying Baculovirus Protein Expression

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Infected cells (

\sim

1.5–2

\times 10^{6}

cells/mL) were collected at

\sim

20–24 hpi by centrifugation at 500

\times g

for 10 min at 4

^{°} C

. The cells were lysed in RIPA buffer (Fisher Scientific), quantified by Pierce BCA assay (Fisher Scientific), and

\sim

μ

g of protein was separated by electrophoresis in 10% TGX Stain-Free precast mini SDS-PAGE gels (Bio-Rad, Mississauga, ON, Canada) according to manufacturer’s directions. After transfer to low fluorescence PVDF membranes, Western blot analysis was performed with anti-GP64 (AcV5, Fisher Scientific) primary antibody and goat anti-mouse IgG HRP secondary (Bio-Rad) and imaged on a ChemiDoc MP Imager (Bio-Rad). The Image Lab software (Bio-Rad) was used for further image processing.

Bruder M.R, & Aucoin M.G. (2023). Evaluation of Virus-Free Manufacture of Recombinant Proteins Using CRISPR-Mediated Gene Disruption in Baculovirus-Infected Insect Cells. Vaccines, 11(2), 225.

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Extracellular Vesicle Protein Profiling

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RBCEVs were lysed with RIPA buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Biotool, USA) for 5 min on ice. Cells were lysed for 30 min on ice. Protein concentration was measured by a Pierce™ BCA assay (Life Technologies, USA) with BSA (New England Biolabs, UK) concentration as standards. A total of 50 μg protein lysate was loaded onto 10% polyacrylamide gels together with a Precision Plus Protein™ Kaleidoscope™ prestained protein standard (Bio‐Rad, USA). The proteins were transferred to Immobilon‐P polyvinylidene difluoride membranes (Merck Millipore, USA), which were blocked using 5% milk in Tris buffered saline containing 0.1% Tween‐20 (TBST) for 1 h followed by an incubation with primary antibody, anti‐ALIX antibody (Santa Cruz, USA, dilution 1:1,000), anti‐TSG101 antibody (Santa Cruz, dilution 1:1,000), anti‐human GAPDH antibody (Santa Cruz, dilution 1:1,000), anti‐human GPA antibody (Santa Cruz, dilution 1:500), anti‐human HBA antibody (Santa Cruz, dilution 1:1,000) or anti‐human β‐actin antibody (CST, USA, dilution 1:1,000) overnight at 4°C. The membranes were washed three times with TBST then incubated with HRP‐conjugated secondary antibody (Jackson ImmunoResearch, USA, dilution 1:5,000) for 1 h at room temperature. The blots were imaged using a ChemiDoc™ gel documentation system (Bio‐Rad).

Peng B., Nguyen T.M., Jayasinghe M.K., Gao C., Pham T.T., Vu L.T., Yeo E.Y., Yap G., Wang L., Goh B.C., Tam W.L., Luo D, & Le M.T. (2022). Robust delivery of RIG‐I agonists using extracellular vesicles for anti‐cancer immunotherapy. Journal of Extracellular Vesicles, 11(4), e12187.

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Protein Quantification by BCA Assay

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Protein concentration was measured using a Pierce BCA assay (Life Technologies, USA) with BSA (NEB, UK) concentrations as standards. A total of 5 µL of samples were loaded into each well of a 96-well plate and 100 µL of BCA solution was added. The plate was incubated at 37°C for 30 min and the absorbance was detected at 562 nm using the Synergy H1 microplate reader (Biotek, USA).

Vu L.T., Peng B., Zhang D.X., Ma V., Mathey-Andrews C.A., Lam C.K., Kiomourtzis T., Jin J., McReynolds L., Huang L., Grimson A., Cho W.C., Lieberman J, & Le M.T. (2019). Tumor-secreted extracellular vesicles promote the activation of cancer-associated fibroblasts via the transfer of microRNA-125b. Journal of Extracellular Vesicles, 8(1), 1599680.

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Western Blot Protein Detection

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Protein lysates were prepared by resuspending cells in RIPA lysis and extraction buffer containing HALT protease inhibitor cocktail (Life Technologies; Carlsbad, CA). Protein concentration of lysates was determined using the Pierce BCA assay (Life Technologies; Carlsbad, CA). 30ug of protein was loaded into a 4–15% Mini-PROTEAN TGX Stain-Free Gel (Bio-Rad; Hercules, CA), run at 200 V for 30 min, and subsequently transferred to an Immobilon-FL PVDF membrane (MilliporeSigma; Burlington, MA) at 100 V for 1 h at 4 °C. Blots were blocked for 1 h at room temperature using a 1:1 dilution of Odyssey Blocking Buffer (LI-COR; Lincoln, NE) and PBS. Primary antibody was then added to blocking buffer plus 0.1% Tween-20 and incubated at room temperature for 1 h. Membranes were incubated with secondary antibody in the same blocking buffer plus 0.1% Tween-20 for 1 h in a light protected box to detect signal. Blots were visualized with an Odyssey Infrared Scanner (LI-COR; Lincoln, NE). Representative images were converted to grayscale for figures. Antibodies used are listed in Supplementary Table S9.

Nghiem-Rao T.H., Pfeifer C., Asuncion M., Nord J., Schill D., Pulakanti K., Patel S.B., Cirillo L.A, & Rao S. (2021). Human induced pluripotent stem cell derived hepatocytes provide insights on parenteral nutrition associated cholestasis in the immature liver. Scientific Reports, 11, 12386.

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MICAL-L1 Immunoblot in HeLa Cells

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HeLa cells were lysed in buffer, 20 mM Tris HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 with a protease cocktail inhibitor (Sigma-Aldrich). Solubilized material was recovered by centrifugation at 18.000 g for 15 min at 4°C and supernatants were collected. Protein amounts were determined using the Pierce BCA assay (Life Technologies, PA, USA) and equal quantities of proteins were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose filters. Immunoblots were performed using anti-MICAL-L1 antibodies and enhanced chemiluminescence according to the manufacturer's protocol (Thermo Fisher Scientific, Rockford, IL, USA).

Sikora R., Bun P., Danglot L., Alqabandi M., Bassereau P., Niedergang F., Galli T, & Zahraoui A. (2021). MICAL-L1 is required for cargo protein delivery to the cell surface. Biology Open, 10(6), bio058008.

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