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Colorimetric assay

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Colorimetric assay is a laboratory technique used to quantify the concentration of a specific substance in a sample. It involves the use of a reagent that reacts with the target substance to produce a color change, which is then measured using a spectrophotometer or colorimeter. The intensity of the color change is proportional to the concentration of the substance in the sample.

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Lab products found in correlation

Enhanced chemi luminescence (ecl), by Cytiva (7 mentions) Trypsin, by Cambrex (5 mentions) Gapdh, by Cell Signaling Technology (5 mentions) Gapdh ab, by Santa Cruz Biotechnology (4 mentions) Western lightning plus ecl reagent, by PerkinElmer (4 mentions) Ethylene diamine tetraacetic acid (edta), by Cambrex (3 mentions) Anti fpr1 polyclonal antibody, by Abcam (3 mentions) Protease inhibitor cocktail, by Roche (3 mentions) Restore western blot stripping buffer, by Thermo Fisher Scientific (3 mentions) Immobilon, by Merck Group (3 mentions) Immobilon western hrp substrate, by Merck Group (3 mentions) Complete mini and phosstop solution, by Roche (3 mentions) Batf3 af7437 antibody, by R&D Systems (2 mentions) C jun g4, by Santa Cruz Biotechnology (2 mentions) Junb c11, by Santa Cruz Biotechnology (2 mentions) Mouse monoclonal anti gapdh, by Santa Cruz Biotechnology (2 mentions) Protease inhibitor, by Roche (2 mentions) Odyssey clx imaging system, by LI COR (2 mentions) Sirt1, by Cell Signaling Technology (2 mentions) Gapdh, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Bradford colorimetric assay (32 citations) Colorimetric protein assay (19 citations) Colorimetric DC protein assay (8 citations) DC colorimetric assay (5 citations) Detergent-compatible colorimetric assay (2 citations) Detergent-compatible colorimetric assay kit (2 citations) Bio-Rad Alkaline Phosphatase Immun-Blot® Colorimetric Assay kit (2 citations) Colorimetric assay method (2 citations) Bradford colorimetric protein assay (2 citations) Colorimetric assay kit (2 citations)

75 protocols using colorimetric assay

1

Protein Immunoblotting and Co-immunoprecipitation

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Whole-cell protein lysates were obtained in RIPA buffer. Protein concentrations were estimated by Bio-Rad colorimetric assay. Immunoblotting was performed as previously described by loading 10 μg of protein onto 7.5% PAGE gels (48 (link)). Signals were detected by enhanced chemiluminescence (Pierce) or by Odyssey Infrared Imager (LI-COR Biosciences). Representative blots are shown. Antibodies used are listed in Supplementary Table S7. Coimmunoprecipitations were performed in whole-cell protein lysates from 15 million cells in 500 μL of nondenaturing buffer (150 mmol/L NaCl, 50 mmol/L Tris-pH8, 1% NP-10, 0.25% sodium deoxycholate) and 10 μL of kinase-specific antibodies. After overnight incubation at 4°C, 50 μL of Protein A agarose beads (Invitrogen) were added and incubated for 1 hour at 4°C. Beads were washed 3 times with nondenaturing buffer, and proteins were eluted in Laemmli sample buffer, boiled, and loaded onto PAGE gels.
Sotillo E., Barrett D.M., Black K.L., Bagashev A., Oldridge D., Wu G., Sussman R., Lanauze C., Ruella M., Gazzara M.R., Martinez N.M., Harrington C.T., Chung E.Y., Perazzelli J., Hofmann T.J., Maude S.L., Raman P., Barrera A., Gill S., Lacey S.F., Melenhorst J.J., Allman D., Jacoby E., Fry T., Mackall C., Barash Y., Lynch K.W., Maris J.M., Grupp S.A, & Thomas-Tikhonenko A. (2015). Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy. Cancer discovery, 5(12), 1282-1295.
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2

Protein Extraction from Tissue and Cells

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The tissue was broken in liquid nitrogen, and homogenized in a lysis buffer consisting of 20 mmol/L Tris (pH 7.5), 10 mmol/L NaCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 2 mmol/L benzamidine, 1 mmol/L PMSF, 50 mmol/L NaF, 1 mmol/L DTT and 10 μg/mL of each leupeptin, pepstatin and aprotinin as previously described [32 (link), 37 (link)]. The tissue homogenates were centrifuged at 2000 rpm in +4 °C for 1 min. To separate the total protein fraction, 5× NEB was added to the tissue homogenate, following by centrifugation at 12,500 rpm for 20 min. The supernatant was stored in −70 °C until assayed.
To extract total protein from cultured cardiomyocytes, the cells were scraped in 100–150 μl of 1× NEB buffer containing protease-inhibitor cocktail (1:100 volume), phosphatase-inhibitor cocktail (1:100 volume) and 1 mM dithiothreitol (DTT) (1:1000 volume). Cells were vortexed for 20 s and centrifuged 12,500 rpm for 20 min at +4 °C. The supernatant was collected as the total protein fragment. The entire procedure was carried out at +4 °C. The supernatant was stored in −70 °C until assayed. Protein concentrations were determined by colorimetric assay (Bio-Rad Laboratories, Hercules, CA, USA).
Kelloniemi A., Aro J., Näpänkangas J., Koivisto E., Mustonen E., Ruskoaho H, & Rysä J. (2015). TSC-22 up-regulates collagen 3a1 gene expression in the rat heart. BMC Cardiovascular Disorders, 15, 122.
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3

Western Blot Analysis of uPAR and FPR1

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Cells detached using 200 mg/L EDTA, 500 mg/L trypsin (Cambrex), were lysed in RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% NP40) containing protease inhibitor mixture. Protein content of cell lysates was measured by a colorimetric assay (BioRad). Twenty and forty µicrograms of proteins from each cell lysate were separated on 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% non-fat dry milk and probed with 1 μg/mL R4 anti-uPAR monoclonal antibody recognizing uPAR D3 domain, 1 μg/mL anti-FPR1 polyclonal antibody (Abcam), or 0.2 μg/mL GAPDH Ab (Santa Cruz Biotechnology). Washed filters were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody and detected by ECL (Amersham-GE Healthcare). Densitometry was performed using the NIH Image 1.62 software (Bethesda, MD). Each experiment was performed three times.
Carriero M.V., Bifulco K., Ingangi V., Costantini S., Botti G., Ragone C., Minopoli M., Motti M.L., Rea D., Scognamiglio G., Botti G., Arra C., Ciliberto G, & Pessi A. (2017). Retro-inverso Urokinase Receptor Antagonists for the Treatment of Metastatic Sarcomas. Scientific Reports, 7, 1312.
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4

Western Blot Analysis of Metabolic Regulators

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Mouse liver, skeletal muscle or white adipose tissue were dissected and immediately frozen in liquid nitrogen. Whole-cell extracts were prepared using lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.2mM Na3VO4 and a protease inhibitor cocktail (Roche Diagnostics)). Protein concentration was measured by colorimetric assay (Bio-Rad Laboratories, Richmond, CA, USA) and equal amount of proteins was loaded onto SDS gels. After transfer to polyvinylidene difluoride membranes, proteins were probed with primary antibodies (1 μg/ml), followed by horseradish peroxidase-conjugated secondary antibodies, washed and visualized with SuperSignal West Pico/Dura chemiluminescent substrate (Pierce-Thermo Fisher Scientific, Waltham, MA, USA). Blots were reprobed with β-actin-specific antibody for loading controls.
Anti-IRS-1 (Santa Cruz Biotechnology), anti-IRS-2 (Santa Cruz Biotechnology), anti-SCD-1 (Santa Cruz Biotechnology), anti-IRβ (Santa Cruz Biotechnology), anti-pAkt2 (Cell Signaling Technology, Danvers, MA, USA), anti-SREBP-1 (Santa Cruz Biotechnology), anti-ACCα (Santa Cruz Biotechnology), anti-FAS (Santa Cruz Biotechnology), anti-SREBP-2 (Santa Cruz Biotechnology), anti-HMGCR (Santa Cruz Biotechnology), anti-Albumin (Santa Cruz Biotechnology) and anti-β-actin (Santa Cruz Biotechnology) antibodies were used as primary antibodies.
Huo J., Ma Y., Liu J.J., Ho Y.S., Liu S., Soh L.Y., Chen S., Xu S., Han W., Hong A., Lim S.C, & Lam K.P. (2016). Loss of Fas apoptosis inhibitory molecule leads to spontaneous obesity and hepatosteatosis. Cell Death & Disease, 7(2), e2091-.
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5

Neutrophil and Inflammatory Biomarker Assays

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Animals were terminated by exsanguination. Lavage fluid neutrophils were evaluated by differential cytology on CytoSpin slides. Lavage protein was determined using a colorimetric assay (Bio-Rad, Hemel Hempstead, UK). CXCL1, Interleukin-6 (IL-6), and soluble Receptor for Advanced Glycation End-products (sRAGE) in lavage fluid, and plasma soluble TNF receptors were determined by ELISA (R&D systems, Abingdon, UK). Lavage fluid TNF was determined by high sensitivity ELISA (eBioscience, Altrincham, UK). Matrix metalloproteinase (MMP) activity was determined by fluorometric assay (abcam, Cambridge, UK). Other plasma biomarkers were determined by FlowCytomix assay (eBioscience).
Wilson M.R., Petrie J.E., Shaw M.W., Hu C., Oakley C.M., Woods S.J., Patel B.V., O’Dea K.P, & Takata M. (2017). High fat feeding protects mice from ventilator-induced lung injury, via neutrophil-independent mechanisms. Critical care medicine, 45(8), e831-e839.
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6

Quantifying PGE2 Production in Cells

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PGE2 formation was quantified in supernatants using a competitive enzyme immunoassay (Cayman, Ann Arbor, USA) with indicated sensitivity of 50 pg/mL and limit of detection at 15 pg/mL. Data are related to the protein content of the corresponding cells which was determined by employing the Lowry method using bovine gamma-globulin as standard [19] (link). The colorimetric assay was purchased from Bio-Rad (München, Germany).
Jaudszus A., Degen C., Barth S.W., Klempt M., Schlörmann W., Roth A., Rohrer C., Sauerwein H., Sachse K, & Jahreis G. (2014). Loss of FADS2 Function Severely Impairs the Use of HeLa Cells as an In Vitro Model for Host Response Studies Involving Fatty Acid Effects. PLoS ONE, 9(12), e115610.
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7

Multiplex Immunomodulatory Protein Assay

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BAL cell pellets were re-suspended in cell lysis buffer (BioRad, Hercules, CA), sonicated for 20 seconds, and passed through a 1.2 μm filter before total protein concentration was determined by colorimetric assay (BioRad). The levels of 27 immunomodulating proteins were then assayed in BAL cell lysates using Bio-Rad Multiplex bead array following manufacturer guidelines as previously described (Bird et al., 2010 (link)). All samples were assayed in duplicate and the results were analyzed using the Bio-Plex manager software, version 5.0. All values were normalized to total protein concentration and reported as picograms of protein of interest per milligram of total protein.
O’Halloran E.B., Curtis B.J., Afshar M., Chen M.M., Kovacs E.J, & Burnham E.L. (2015). Alveolar macrophage inflammatory mediator expression is elevated in the setting of alcohol use disorders. Alcohol (Fayetteville, N.Y.), 50, 43-50.
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8

Immunoblotting and Immunoprecipitation of Transcription Factors

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Whole-cell protein lysates were obtained in nondenaturing buffer (150 mmol/L NaCl, 50 mmol/L Tris-pH8, 1% NP-10, 0.25% sodium deoxycholate). Protein concentrations were estimated by Bio-Rad colorimetric assay. Immunoblotting was performed by loading 20μg of protein onto 11% PAGE gels followed by transfer to PVF membranes. Signals were detected by enhanced chemiluminescence (Pierce) or with the Odyssey imaging system. Representative blots are shown. The following primary antibodies used were purchased from Cell Signaling: c-Jun (60A8), P-c-JunSer73 (D47G9), JunB(C37F9), BATF(D7C5), IRF4(4964) and Histone-3(1B1B2). The BATF3 (AF7437) antibody was from R&D. Immunoprecipitations were performed in 100mg of whole-cell protein lysates in 150μL of nondenaturing buffer and 7.5mg of agar-conjugated antibodies c-Jun (G4) or JunB (C11) (Santa Cruz Biotechnology). After overnight incubation at 4°C. Beads were washed 3 times with nondenaturing buffer, and proteins were eluted in Laemmli sample buffer, boiled, and loaded onto PAGE gels. Detection of immunoprecipitated proteins was performed with above-mentioned reagents and antibodies.
Lynn R.C., Weber E.W., Sotillo E., Gennert D., Xu P., Good Z., Anbunathan H., Lattin J., Jones R., Tieu V., Nagaraja S., Granja J., DeBourcy C., Majzner R., Satpathy A.T., Quake S.R., Monje M., Chang H, & Mackall C.L. (2019). c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature, 576(7786), 293-300.
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9

CD3-zeta Immunoblotting in Cell Lysates

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Whole-cell protein lysates were obtained in non-denaturing buffer as previously described(Lynn et al., 2019 (link)). Protein concentrations were estimated by Bio-Rad colorimetric assay. Immunoblotting was performed by loading 10 μg of protein onto 7.5% PAGE gels followed by transfer to PVF membranes. Signals were detected by enhanced chemiluminescence (Pierce) or with the Odyssey imaging system. The CD3-zeta (4A12-F6) primary antibody was purchased from Abcam. Total ERK1/2 (#9102) and GAPDH (#97166) antibodies were purchased from Cell Signaling and used as loading control. Images were cropped to exclude non-relevant gel lanes.
Labanieh L., Majzner R.G., Klysz D., Sotillo E., Fisher C.J., Vilches-Moure J.G., Pacheco K.Z., Malipatlolla M., Xu P., Hui J.H., Murty T., Theruvath J., Mehta N., Yamada-Hunter S.A., Weber E.W., Heitzeneder S., Parker K.R., Satpathy A.T., Chang H.Y., Lin M.Z., Cochran J.R, & Mackall C.L. (2022). Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell, 185(10), 1745-1763.e22.
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10

Western Blotting of IVC Proteins

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The entire IVC was removed with care taken to avoid surrounding arterial and connective tissues. Proteins were extracted with RIPA lysis buffer containing protease inhibitors from single IVC.11 (link), 13 (link), 21 (link) Protein concentrations were assessed using a colorimetric assay (Bio Rad), and equal amounts of protein were run on a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and then transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk, membranes were probed overnight with the primary antibodies (Major Resources Table). After overnight incubation, the membranes were incubated with HRP conjugated secondary antibodies for 1 hour at room temperature and were developed with using Western Lightning Plus ECL reagent (NEL105001EA, PerkinElmer; Waltham, MA).
Matsubara Y., Gonzalez L., Kiwan G., Liu J., Langford J., Gao M., Gao X., Taniguchi R., Yatsula B., Furuyama T., Matsumoto T., Komori K., Mori M, & Dardik A. (2021). PD-L1 regulates T-cell differentiation to control adaptive venous remodeling. Arteriosclerosis, thrombosis, and vascular biology, 41(12), 2909-2922.
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