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Modified lowry assay

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The Modified Lowry assay is a colorimetric method used to quantify the total protein content in a sample. It is a modification of the original Lowry method, which measures the reduction of the Folin-Ciocalteu reagent by aromatic amino acid residues in the sample. The modified version improves the assay's sensitivity and reproducibility, making it a widely used technique in various fields of research and analysis.

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Lab products found in correlation

Image lab software, by Bio-Rad (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Gel doc ez imager, by Bio-Rad (2 mentions) Ne per protocol, by Thermo Fisher Scientific (2 mentions) Tbs 1 casein, by Bio-Rad (2 mentions) Phosphatase inhibitor cocktail p5726, by Merck Group (1 mentions) Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions) Symmetry c18 resin, by Waters Corporation (1 mentions) Nanoacquity uplc, by Waters Corporation (1 mentions) Beh130 c18 column, by Waters Corporation (1 mentions) Power blotter semi dry transfer system, by Thermo Fisher Scientific (1 mentions) Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions) Microfuge 22r centrifuge, by Beckman Coulter (1 mentions)

Spelling variants of this product

Modified Lowry Protein Assay Kit (31 citations) Pierce™ Modified Lowry Protein Assay Kit (20 citations) Modified Lowry Protein Assay (9 citations)

6 protocols using modified lowry assay

1

Analyzing NF-kB Nuclear Translocation

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To assess nuclear translocation of NF-kB, cells were lysed after 20 h using the NE-PER protocol (Thermo Scientific). Using this kit, the cytoplasmic and nuclear protein fractions are separated from each other. To asses IκBα expression, cells were lysed with RIPA buffer. Protein content of the lysates was determined using the modified Lowry assay (Thermo Scientific). Proteins (20 µg) were separated using an Any kD gel (SDS-PAGE) and transferred to a nitrocellulose membrane (BioRad, Temse, Belgium). Aspecific binding sites were blocked for 30 min using TBS + 1% casein (BioRad, Temse, Belgium). Western blot was performed using the following primary polyclonal antibodies overnight (4 °C): anti-p65 NFkB (1/500), anti-IκBα (1/1000) and anti-β-actin (1/1000), β-tubulin (1/4000) and anti-Histon H3.3 (1/5000) were used as loading controls. Goat anti-rabbit-AP antibody was used for detection (1/10,000) (60 min). All antibodies were purchased at Thermo Scientific (Merelbeke, Belgium). Finally, the BCIP/NBT substrate was added and the results were analyzed using the GelDoc EZ imager and Image Lab software (BioRad, Temse, Belgium). TBS buffer was used for washing between the steps.
Janssens Y., Debunne N., De Spiegeleer A., Wynendaele E., Planas M., Feliu L., Quarta A., Claes C., Van Dam D., De Deyn P.P., Ponsaerts P., Blurton-Jones M, & De Spiegeleer B. (2021). PapRIV, a BV-2 microglial cell activating quorum sensing peptide. Scientific Reports, 11, 10723.
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2

Protein Extraction and Quantification

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Protein was extracted using radio immune precipitation (RIPA) lysis buffer complemented with a Protease inhibitor cocktail (Set V, Calbiochem, Dorset, UK) and a phosphatase inhibitor cocktail (P5726 (Sigma, Dorset, UK)). Cell lysates were vortexed every 2 min and kept on ice for 10 min before being centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatants were collected and total proteins were quantified using a Modified Lowry assay (Thermo Scientific, Oxford, UK). All experiments were done in 3 biological and experimental repeats.
Tejpal S., Wemyss A.M., Bastie C.C, & Klein-Seetharaman J. (2020). Lemon Extract Reduces Angiotensin Converting Enzyme (ACE) Expression and Activity and Increases Insulin Sensitivity and Lipolysis in Mouse Adipocytes. Nutrients, 12(8), 2348.
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3

Quantitative Shotgun Proteomics by LC-MS/MS

Cited 2 times
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LC-MS/MS analysis was carried out using a nano-ACQUITY UPLC (Waters, Milford, MA) that was interfaced with an LTQ-Orbitrap XL (Thermo Scientific, Waltham, MA) mass spectrometer. For each analysis, 2 µl of tryptic digest containing an estimated 1.0 µg of digested peptides was loaded onto a 180 µm × 20 mm trap column packed with 5 µm Symmetry C18 resin (Waters, Milford, MA) using solvent A (0.1% formic acid in Milli-Q water) for 5 min, followed by separation in a 75 µm×250 mm analytical 1.7 µm BEH130 C18 column (Waters, Milford, MA) using an 240 min gradient with solvent B (0.1% formic acid in acetonitrile) [41 (link), 42 (link)]. Peptide amounts in digests were estimated based on analysis of total protein measured by the modified Lowry assay (Thermo Scientific, Rockford, IL) and assuming an approximate recovery of 25% after trypsin digestion as indicated by similar analysis of standard protein samples. A 25 min blank gradient was run between each sample injection to minimize peptide carryover. Full scans were carried out from 400 to 2000 m/z with 60,000 resolution. MS2 data were acquired through data-dependent analysis of the top six most intense ions with dynamic exclusion enabled for 60 s, monoisotopic precursor selection enabled, and single charged ions rejected.
Basu A., Harper S., Pesciotta E.N., Speicher K.D., Chakrabarti A, & Speicher D.W. (2015). Proteome Analysis of the Triton-Insoluble Erythrocyte Membrane Skeleton. Journal of proteomics, 128, 298-305.
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4

Quantitative Western Blot Analysis of C. difficile Toxins

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Protein concentrations were determined using a modified Lowry assay (Thermo Scientific, Waltham, MA) with bovine serum albumin as a standard. Equal amounts of protein (40 μg) per sample were combined with SDS-loading buffer (2X Laemmli buffer) with 5% 2-Mercaptoethanol, and run on pre-casted 7.5% acrylamide gels (Bio-Rad, Hercules, CA) at 100V for 90 min. For immunoblots performed with recombinant protein a total of 50 ng was used of CI_01447 and CI_01448 was used. For C. difficile TcdA and TcdB, 1 ng was used. Proteins transferred onto polyvinylidene difluoride (PVDF) membranes using a Power Blotter–Semi-dry Transfer System (Thermo Fisher Scientific, Waltham, MA). Membranes were incubated in blocking buffer (5% Skim milk, 0.1% Tween 20 in PBS) for 2 h at room temperature or overnight at 4°C with gentle shaking. Blots were probed with a 1: 5,000 dilution of chicken Clostridium difficile anti-toxin A, conjugated with HRP (ACdTA-HRP), or with chicken Clostridium difficile anti-toxin B, conjugated with HRP (AcdTB-HRP) (Exalpha Biologicals, Inc., Shirley, MA). Blots were subsequently developed using Immun-Star WesternC chemiluminescence reagents (Bio-Rad, Hercules, CA) and imaged using Syn G: BOX Chemi XX6 (Syngene International Limited, India).
Cherny K., Balaji A., Mukherjee J., Goo Y., Hauser A., Ozer E., Satchell K., Bachta K., Kochan T., Mitra S, & Kociolek L. (2022). Identification of Clostridium innocuum hypothetical protein that is cross-reactive with C. difficile anti-toxin antibodies. Anaerobe, 75, 102555.
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5

Evaluating NF-kB Nuclear Translocation

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To assess nuclear translocation of NF-kB, cells were lysed after 20h using the NE-PER™ protocol (Thermo Scientific). Using this kit, the cytoplasmic and nuclear protein fractions are separated from each other. To asses IκBα expression, cells were lysed with RIPA buffer. Protein content of the lysates was determined using the modified Lowry assay (Thermo Scientific).
Proteins (20 µg) were separated using an Any kD gel (SDS-PAGE) and transferred to a nitrocellulose membrane (BioRad, Temse, Belgium). Aspecific binding sites were blocked for 30 min using TBS + 1% casein (BioRad, Temse, Belgium). Western blot was performed using the following primary antibodies overnight (4°C): anti-p65 NFkB (1/500), anti-IκBα (1/1000) and anti-β-actin (1/1000), β-tubulin (1/4000) and anti-Histon H3.3 (1/5000) were used as loading controls. Goat anti-rabbit-AP antibody was used for detection (1/10000) (60 min). All antibodies were purchased at Thermo Scientific (Merelbeke, Belgium). Finally, the BCIP/NBT substrate was added and the results were analyzed using the GelDoc EZ imager and Image Lab software (BioRad, Temse, Belgium). TBS buffer was used for washing between the steps.
Janssens Y., Debunne N., Spiegeleer A.D., Wynendaele E., Planas M., Feliu L., Quarta A., Claes C., Dam D.V., Deyn P.P.D., Ponsaerts P., Blurton‐Jones M., & Spiegeleer B.D. (2020). PapRIV, a BV-2 microglial cell activating quorum sensing peptide.
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6

Dentin Matrix Metalloproteinase Extraction

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Isolated dentin powder was transferred to a 1.5 mL microcentrifuge tube and partially demineralized with 1 mL of 1% phosphoric acid (H 3PO4) for 10 min with rotation at 4°C to increase exposure of dentin powder to the acid. All specimens were then rinsed 3 times with 1 mL of distilled water. The demineralized dentin powder was resuspended in different volumes and compositions of MMP extraction buffer (MEB; Table 1) and again rotated at 4°C for 18 h after ultrasonication with the Virsonic 50 for 3 spurts of 10 s (VirTis, Gardiner, NY, USA). The vials were centrifuged at 13,000 rpm (15,000 rcf) for 30 min at 4°C in a Beckman-Coulter Microfuge 22R Centrifuge, and the supernatants were collected. The supernatants were dialyzed through a 30-kDa cutoff membrane (Slide-A-Lyzer Dialysis Cassettes, Thermo Scientific, Rockford, IL, USA) against extraction buffer overnight. The dialyzed samples were concentrated 3× by Amicon Ultra-4 at 3,000 rpm for 10 min in a Beckman Allegra 6 centrifuge (EMD Millipore, Bellerica, MA, USA). Total protein concentration of dentin extracts was determined using Modified Lowry Assay (Thermo Scientific).
Kang J., Izutani N., D'Angelo M., Buis W., Wang Y., Blatz M., Imazato S, & Ozer F. (2019). Assaying endogenous matrix metalloproteinases (MMPs) in acid-etched dentinal cavity walls. Dental materials journal, 38(6).
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