Pierce micro bca protein assay

Manufactured by Thermo Fisher Scientific

Sourced in United States, Australia

The Pierce Micro BCA Protein Assay is a colorimetric detection and quantitation method for measuring the total protein concentration in a sample. It is based on the bicinchoninic acid (BCA) reaction, in which peptide bonds in protein reduce Cu2+ to Cu+ in an alkaline environment. The purple-colored reaction product formed by the chelation of two molecules of BCA with one cuprous ion is measured spectrophotometrically at 562 nm, providing a basis to quantify the total protein present in the sample.

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Lab products found in correlation

Gpo pap reagent, by Roche (2 mentions) Hitrap, by Cytiva (2 mentions) Amplex red cholesterol kit, by Thermo Fisher Scientific (1 mentions) Free glycerol reagent, by Merck Group (1 mentions) Nefa c, by Fujifilm (1 mentions) Rabbit anti gapdh, by Merck Group (1 mentions) Recombinant human pdgf bb, by Thermo Fisher Scientific (1 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Phosphatase inhibitor, by Merck Group (1 mentions) Rabbit anti akt 9272, by Cell Signaling Technology (1 mentions) Ripa buffer, by Merck Group (1 mentions) Chloroform, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Biowest (1 mentions) Gradient polyacrylamide gel, by Bio-Rad (1 mentions) Pdgf bb elisa kit, by Thermo Fisher Scientific (1 mentions) Rabbit anti p akt ser473, by Cell Signaling Technology (1 mentions) Amphotericin b, by Biowest (1 mentions) Gentamicin, by Biowest (1 mentions) 2 mercaptoethanol, by Merck Group (1 mentions) Laemmli protein sample buffer, by Bio-Rad (1 mentions)

Spelling variants of this product

BCA assay (3195 citations) BCA protein assay (2173 citations) Pierce BCA assay (329 citations) Micro BCA assay (172 citations) Pierce BCA (78 citations) Protein assay (17 citations) Pierce Micro BCA (4 citations) Pierce Micro BCATM Protein Assay (2 citations) Pierce Micro BCATM Protein Assay Kit (2 citations)

15 protocols using pierce micro bca protein assay

Cellular Lipid Extraction and Cholesterol Analysis

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Cellular lipids were extracted using the method of Folch et al. [22 (link)] and cholesteryl esters were quantified using an enzymatic Amplex Red® Cholesterol kit (Thermo Fisher Scientific) [23 (link)]. Cell protein content was determined using Pierce Micro BCA protein assay (Life Technologies Australia Pty Ltd., Scoresby VIC, Australia). Media cholesterol was measured using the Amplex Red® Cholesterol kit according to manufacturer’s instructions (Thermo Fisher Scientific) [23 (link)]. Media testosterone was determined by the Department of Chemical Pathology, Royal Prince Alfred Hospital (Sydney, NSW, Australia). Neutral cholesterol ester hydrolase activity was determined as previously described [24 (link)].

Raftopulos N.L., Washaya T.C., Niederprüm A., Egert A., Hakeem-Sanni M.F., Varney B., Aishah A., Georgieva M.L., Olsson E., dos Santos D.Z., Nassar Z.D., Cochran B.J., Nagarajan S.R., Kakani M.S., Hastings J.F., Croucher D.R., Rye K.A., Butler L.M., Grewal T, & Hoy A.J. (2022). Prostate cancer cell proliferation is influenced by LDL-cholesterol availability and cholesteryl ester turnover. Cancer & Metabolism, 10, 1.

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Measuring Adipocyte Lipid Profiles

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Concentration of non-esterified fatty acids (NEFA-C, WAKO Diagnostics, Richmond, VA, USA) and glycerol (Free glycerol reagent, Sigma-Aldrich, Castle Hill, NSW, Australia) was determined using commercial kits. Adipocyte triacylglycerol (TAG) content was extracted using the method of Folch et al. [26 (link)] and quantified using an enzymatic colorimetric method (GPO-PAP reagent, Roche Diagnostics). Cell protein content was determined using Pierce Micro BCA protein assay (Life Technologies Australia Pty Ltd., Scoresby VIC, Australia).

Balaban S., Shearer R.F., Lee L.S., van Geldermalsen M., Schreuder M., Shtein H.C., Cairns R., Thomas K.C., Fazakerley D.J., Grewal T., Holst J., Saunders D.N, & Hoy A.J. (2017). Adipocyte lipolysis links obesity to breast cancer growth: adipocyte-derived fatty acids drive breast cancer cell proliferation and migration. Cancer & Metabolism, 5, 1.

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Folch Extraction and Quantification

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Cell TAG were extracted using the method of Folch et al. (1957) and quantified using an enzymatic colorimetric method (GPO‐PAP reagent, Roche Diagnostics, North Ryde, NSW, Australia). Cell protein content was determined using Pierce Micro BCA protein assay (Life Technologies Australia Pty Ltd.).

Balaban S., Lee L.S., Varney B., Aishah A., Gao Q., Shearer R.F., Saunders D.N., Grewal T, & Hoy A.J. (2018). Heterogeneity of fatty acid metabolism in breast cancer cells underlies differential sensitivity to palmitate‐induced apoptosis. Molecular Oncology, 12(9), 1623-1638.

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Biodegradable Polyester Urethane Copolymer Characterization

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A biodegradable polyester urethane block copolymer, commercially available as DegraPol15 (DP) (M_w = 65 kDa) was kindly provided Ab Medica, Italy. The polymer was produced according to the procedure described earlier^{25 ,44}. Chloroform, ≥99.8% (0.5–1.0% ethanol as stabilized), 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, ≥95%, RPMI vitamins solution, RIPA buffer, phosphatase inhibitors and 0.5 M EDTA solution were purchased from Sigma-Aldrich, Switzerland. Recombinant human PDGF-BB and PDGF-BB ELISA kit were purchased from PeproTech. Ham’s F12 cell culture media, gentamicin, amphotericin B and Fetal Bovine Serum (FBS) were bought from Biowest, while non-essential amino acids solution, Pierce micro BCA protein assay and Pierce ECL Western Blotting Substrate were purchased from Thermo Scientific. Primaria^TM tissue culture plates were purchased from Corning. Primary antibodies used for western blot included rabbit anti Akt (9272 S, Cell Signaling Technology), rabbit anti pAkt (Ser473) (4060 S, Cell Signaling Technology) and rabbit anti GAPDH (G9545, Sigma Aldrich). Donkey anti rabbit antibody conjugated with HRP (Jackson) was used as secondary antibody for western blot. 4–20% gradient polyacrylamide gels were purchased from Bio-Rad, Switzerland.

Evrova O., Kellenberger D., Scalera C., Calcagni M., Giovanoli P., Vogel V, & Buschmann J. (2019). Impact of UV sterilization and short term storage on the in vitro release kinetics and bioactivity of biomolecules from electrospun scaffolds. Scientific Reports, 9, 15117.

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Protein Fractionation of Rat DLS and DMS

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Rats used in the 0 vs. 120 cue extinction experiment were killed by decapitation immediately after the cue-induced drug seeking test. Brains were flash-frozen in isopentane on dry ice and kept at −80 °C. The anterior DLS (AP +2 mm to +0.8 mm) and posterior DMS (AP +1 mm to −0.4 mm) were dissected over dry ice from coronal sections made ~1 mm thick on a stainless steel brain matrix (Braintree Scientific). We chose to focus our analysis on the anterior portion of the DLS and the posterior portion of the DMS based on previous publications suggesting this distinction is important (Furlong et al., 2018 (link); Yin et al., 2005 (link)). Tissue was fractionated into membrane- and non-membrane-bound components as previously described (Bañuelos et al., 2014 (link)). Protein concentrations for each sample were determined using a Thermo Scientific Pierce Micro BCA Protein Assay (Thermo Fischer Scientific) as previously described (Kirschmann et al., 2017 (link)). For each sample, 20 μg of protein was diluted in 30 μl containing 8 μl of sample buffer (a 9:1 mixture of 4x Laemmli protein sample buffer [Bio-Rad] to 2-Mercaptoethanol [Millipore Sigma]).

Bender B.N, & Torregrossa M.M. (2020). Dorsolateral striatum dopamine-dependent cocaine seeking is resistant to Pavlovian cue extinction in male and female rats. Neuropharmacology, 182, 108403.

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Aβ1–42 Oligomer Preparation Protocol

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Aβ_1–42 oligomers (AβO) were prepared as previously described (Brkic et al, 2015b). Briefly, Aβ_1–42 (rPeptide; A‐1163‐1) or scrambled peptide (rPeptide; A‐1004‐1) was dissolved in hexafluoroisopropanol (HFIP; Sigma‐Aldrich; 105228) at 1 mg/ml. Next, HFIP was removed with a SpeedVac vacuum concentrator. The resulting peptides were then dissolved in DMSO and purified using a 5 ml HiTrap desalting column (GE Healthcare; 17‐408‐01). The monomeric Aβ_1–42 was eluted with Tris‐EDTA buffer (50 mM Tris and 1 mM EDTA, pH 7.5). The eluted peptide concentration was measured using Pierce Micro BCA Protein Assay (Thermo Scientific; 23225) according to the manufacturer's guidelines. The peptides were allowed to oligomerize at room temperature (RT) for 2 h, followed by dilution to 1 μg/ml or 2 μg/ml in Tris‐EDTA buffer. All icv injections were performed within 2 h after oligomerization.

Steeland S., Gorlé N., Vandendriessche C., Balusu S., Brkic M., Van Cauwenberghe C., Van Imschoot G., Van Wonterghem E., De Rycke R., Kremer A., Lippens S., Stopa E., Johanson C.E., Libert C, & Vandenbroucke R.E. (2018). Counteracting the effects of TNF receptor‐1 has therapeutic potential in Alzheimer's disease. EMBO Molecular Medicine, 10(4), e8300.

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Quantification of Stratum Corneum Sphingolipids

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Sphingoid bases were separated by RP-UPLC using an Acquity I-Class UPLC with BEH C18 column, 2.1 × 50 mm with 1.7 μm particle size (Waters Inc.) and detected by electron spray ionisation in positive mode and MS/MS-instrument (Xevo TQ MS, Waters Inc., Milford, MA, U.S.A.) in multiple reaction monitoring (MRM) mode (see Supplementary Table S1) according to the method described previously [49 (link)]. Details on MS analysis are shown in Supplementary Table S1.
To compensate for variable amount of stratum corneum on the tape, the concentrations of all analytes were normalized for protein content on the tape [47 (link)]. For that purpose, 150 µl of the extract obtained after the extraction with methanol:chloroform:water was evaporated to dryness under a stream of nitrogen, and to the residue 250 µl water was added. The protein amount was determined by Pierce Micro BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA).

Jurakic Toncic R., Jakasa I., Ljubojevic Hadzavdic S., Goorden S.M., Ghauharali-van der Vlugt K.J., Stet F.S., Balic A., Petkovic M., Pavicic B., Zuzul K., Marinovic B, & Kezic S. (2020). Altered Levels of Sphingosine, Sphinganine and Their Ceramides in Atopic Dermatitis Are Related to Skin Barrier Function, Disease Severity and Local Cytokine Milieu. International Journal of Molecular Sciences, 21(6), 1958.

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Quantitative Assessment of Protein Expression

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Cell lysates or conditioned media were collected after a specified incubation duration. Total protein amounts in the cell lysates were determined using the Pierce Micro BCA Protein Assay (Thermo Scientific) and prepared as previously described (Li et al., 2010 (link)). The conditioned media were concentrated using VivaSpin 500 concentrators (Sartorius Stedim Biotech GmbH) and the same amount of volume per sample was loaded. The cell lysates and conditioned media were assayed as previously described, but briefly, equal amounts of protein or volume were loaded in gelatin embedded polyacrylamide gels to separate the protein using SDS-PAGE techniques (Wilder et al., 2011 (link)). The gel was washed in renaturing buffer and assay buffer followed by staining with a Coomassie blue stain and destain. The gel was then imaged using an ImageQuant LAS 4000 (GE Healthcare Life Sciences). The bands were then quantified using ImageJ.

Wilder C.L., Walton C., Watson V., Stewart F.A., Johnson J., Peyton S.R., Payne C.K., Odero-Marah V, & Platt M.O. (2016). Differential cathepsin responses to inhibitor-induced feedback: E-64 and cystatin C elevate active cathepsin S and suppress active cathepsin L in breast cancer cells. The international journal of biochemistry & cell biology, 79, 199-208.

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EV Lysis and Protein Quantification

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EVs were lysed by the addition of a RIPA buffer at a ratio of 1:1, incubated on ice for 25 min and centrifuged at 10,000× g for 10 min at 4 °C. Total protein concentration was determined using Pierce MicroBCA Protein Assay following manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA).

Newman L.A., Useckaite Z., Johnson J., Sorich M.J., Hopkins A.M, & Rowland A. (2022). Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease. Biomedicines, 10(1), 195.

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Generation and Purification of Phosphorylated Alpha-Synuclein

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Recombinant human full length, 119CTT and 122CTT aSyn proteoforms were generated according to the protocol previously published by our and other groups [18 (link), 38 ]. Polo like kinase 2 (PLK2) was expressed in BL21-DE3-pLysS competent E. coli, isolated via its His-tag and immediately used to phosphorylate purified aSyn. Briefly, aSyn was mixed with PLK2 buffer (200 mM Tris pH 7.5/100 mM MgCl2/10 mM DTT), 10 mM ATP, H₂O and PLK2, in a molar ratio of aSyn to PLK2 of 167:1. After incubation for 24 h at 30 °C without shaking, samples were diluted 1:3 in 20 mM Tris pH 7.5 buffer (buffer A) and loaded on a 1 ml HiTrap Q column (GE Healthcare). After washing with buffer A, the proteins were eluted with a gradient from 20–55% buffer B (20 mM Tris pH 7.5/1 M NaCl) in 35 column volumes. Chromatography was performed on a ÄKTA explorer (GE Healthcare). The fractions containing phosphorylated aSyn were pooled, concentrated to 0.91 mg/ml (measured with Pierce micro BCA protein assay; Thermo Scientific) using a 3kD Amicon Ultra unit, aliquoted and frozen at − 80 °C.

Moors T.E., Mona D., Luehe S., Duran-Pacheco G., Spycher L., Mundigl O., Kaluza K., Huber S., Hug M.N., Kremer T., Ritter M., Dziadek S., Dernick G., van de Berg W.D, & Britschgi M. (2022). Multi-platform quantitation of alpha-synuclein human brain proteoforms suggests disease-specific biochemical profiles of synucleinopathies. Acta Neuropathologica Communications, 10, 82.

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