660 nm protein assay reagent

Manufactured by Thermo Fisher Scientific

The 660 nm Protein Assay Reagent is a colorimetric assay for the determination of protein concentration. It measures the absorbance of the sample at 660 nm.

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Pierce 660 nm Protein Assay Reagent (168 citations) Protein assay reagent (28 citations) Protein assay (17 citations) 660 nm Protein Assay (14 citations) Pierce 660 nm Protein Assay with Ionic detergent compatibility reagent (2 citations) Pierce 660 nm protein assay reagent kit (2 citations) Pierce-660 nm ready-to-use Protein Assay Reagent (2 citations)

7 protocols using 660 nm protein assay reagent

Quantitative Mitochondrial Protein Analysis

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For each specimen, four thoraxes were dissected and homogenized by dounce tissue grinder in RIPA buffer containing protease inhibitor (cOmplete^TM, Roche). Cellular debris were removed by centrifugation at 4 °C, 14000× g for 20 min. The supernatants were collected and the protein concentrations were determined by Pierce protein assay (Pierce 660 nm Protein Assay Reagent, ThermoScientific). 0.6 μg/well of proteins were loaded for SDS-PAGE and western-blot analysis.
The primary antibodies and the dilutions were as follows: mouse anti-ATP5A (50000x, abcam ab14748), mouse anti-Cytochrome C (10000x, abcam ab13575), mouse anti-PDHA1 (1000x, abcam ab110334), rabbit anti-SOD2 (10000x, abcam ab13534) and rabbit anti-alpha Tubulin (10000x, abcam ab18251). The secondary antibodies and the dilutions were as follows: anti-mouse IgG-HRP (2000x, Invitrogen 62–6520), or anti-rabbit IgG-HRP (5000x, abcam ab97051). The signals were developed by chemiluminescent HRP substrate (Immobilon^TM Western, Millipore) and recorded by a digital multispectral imaging system (BioSpectrum, UVP).

Jiang Y.F., Lin S.S., Chen J.M., Tsai H.Z., Hsieh T.S, & Fu C.Y. (2017). Electron tomographic analysis reveals ultrastructural features of mitochondrial cristae architecture which reflect energetic state and aging. Scientific Reports, 7, 45474.

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Western Blot Analysis of Organoid Proteins

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Organoids were recovered from Matrigel with Cell Recovery media (cat no: 354253; Corning). Organoids were washed with PBS and the cells were lysed with 2× Laemmli solution and boiled at 98ºC. Protein concentrations were measured by a Pierce (Waltham, MA) 660 nm Protein Assay Reagent and IDCR (cat no: 22660; ThermoFisher Scientific). Samples were loaded equally in terms of protein concentration into 10% Bis-Tris protein gels (cat no: 4561033; Bio-Rad) and blotted on nitrocellulose membranes. Membranes were incubated with primary antibodies: anti-Esrrg (cat no: ab49129; Abcam); anti–Spi-B (Spi-B D4V9S, cat no: 14337; CST); anti-H3K27me3 (cat no: ab192985; Abcam); anti-H3 (cat no: ab1791; Abcam), anti-Rank (cat no: MBS9133424; MyBioSource, San Diego, CA), and anti-glyceraldehyde-3-phosphate dehydrogenase (cat no: ab8245; Abcam) at 4°C overnight, and horseradish-peroxidase–conjugated anti-rabbit (1:5000, cat no: RABHRP1-10UL; Sigma-Aldrich) or anti-mouse (1:1000, cat no: 7076; CST) for 1 hour at room temperature. Signal was detected using ECL reagent (cat no: 2232; Amersham, Amersham, UK).

George J.J., Oittinen M., Martin-Diaz L., Zapilko V., Iqbal S., Rintakangas T., Arrojo Martins F.T., Niskanen H., Katajisto P., Kaikkonen M.U, & Viiri K. (2021). Polycomb Repressive Complex 2 Regulates Genes Necessary for Intestinal Microfold Cell (M Cell) Development. Cellular and Molecular Gastroenterology and Hepatology, 12(3), 873-889.

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Murine Brain Protein Extraction and Western Blot

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Protein was isolated from liquid nitrogen shock frozen murine brain-hippocampus tissue using N-Per following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA) and included a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Protein was quantified using a 660nm protein assay reagent (Thermo Fisher Scientific, Waltham, MA). 20 μg of protein was denatured at 95°C in Laemmli sample buffer for 5 min. Samples were resolved on a 4–10% polyacrylamide gel and transferred to nitrocellulose membranes, which were blocked for 1h at room temperature in either 3% BSA / TBST. The membranes were incubated in primary antibody overnight at 4°C. The primary antibodies used were rabbit polyclonal PER2 (AB2202, Millipore Sigma, Burlington, MA; antibody verified for PER2 specificity [35 (link)]), mouse monoclonal β-actin (8H10D10, Cell signaling, Danvers, MA), The secondary antibodies used were goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA) and goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA).

Gile J., Oyama Y., Shuff S, & Eckle T. (2020). A role for the adenosine ADORA2B receptor in midazolam induced cognitive dysfunction. Current pharmaceutical design, 26(34), 4330-4337.

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Quantitative Analysis of MYPT1 and RhoA Signaling

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NIH/3T3 cells were rinsed once with PBS and lysed in M-PER lysis buffer (Thermo Fisher) supplemented with protease and phosphatase inhibitor cocktail (Sigma). Protein concentration of the lysates cleared of insoluble cell debris were determined using 660 nm Protein Assay reagent (Thermo Fisher). A total of 15 μg of proteins in LDS electrophoresis loading buffer (Life Technologies) was denatured for 10 min at 70 °C and separated on 4–12% SDS-PAGE gel (Life Technologies). Proteins were transferred onto 0.2-μm nitrocellulose membrane (Pall) and processed for Western blotting. Primary antibodies were used at the following dilutions: goat anti-actin (sc-1616, Santa-Cruz Biotechnology, 1:4,000), mouse anti-MYPT1 (612165, Becton-Dickinson, 1:4,000), rabbit anti-MYPT1(pT696) (ABS45, Millipore, 1:500), rabbit anti-RhoA (67B9, Cell Signaling, 1:4,000), rabbit anti-MLC2 (8505, Cell Signaling, 1:4,000), and mouse anti-MLC2(pT19) (3674, Cell Signaling, 1:500). Appropriate secondary IRDye-conjugated antibodies (LI-COR) were used at 1:10,000. Proteins were detected using Odyssey imager (LI-COR). The investigator carrying out the Western blot experiments was not blinded to the identity of the samples.

Vabres P., Sorlin A., Kholmanskikh S.S., Demeer B., St-Onge J., Duffourd Y., Kuentz P., Courcet J.B., Carmignac V., Garret P., Bessis D., Boute O., Bron A., Captier G., Carmi E., Devauchelle B., Geneviève D., Gondry-Jouet C., Guibaud L., Lafon A., Mathieu-Dramard M., Thevenon J., Dobyns W.B., Bernard G., Polubothu S., Faravelli F., Kinsler V.A., Thauvin C., Faivre L., Ross M.E, & Rivière J.B. (2019). Postzygotic inactivating mutations of RHOA cause a mosaic neuroectodermal syndrome. Nature genetics, 51(10), 1438-1441.

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Western Blot Analysis of Cell Signaling

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Cells were lysed in Nonidet P-40 (NP-40) buffer (1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1X Complete Mini protease inhibitor cocktail [Roche], 1X phosphatase inhibitor cocktail 2 [Sigma] in PBS). Protein concentrations of lysates were determined using 660 nm Protein Assay Reagent (Thermo Scientific), according to the manufacturer’s protocol. Equal amounts of protein were resolved on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dried milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20; pH 7.6) and then incubated in primary antibody. Following primary antibody incubation, membranes were washed three times with 1% non-fat dried milk in TBS-T, incubated in secondary antibody, and then washed three more times with 1% non-fat dried milk in TBS-T. Proteins were visualized by incubating membranes in ECL chemicals and exposing to film. Bands were quantified using ImageJ software. The following primary antibodies were used for western blotting: 4E-BP1, β-tubulin, cyclin D1, eIF2α, eIF4E, eIF4G, GCN2, Ku80, p27, p-4E-BP1, p-eEF2, p-eIF2α, p-p53 (all from Cell Signaling); β-actin (Sigma), and p21 (BD Pharmigen). The secondary antibodies used were anti-rabbit HRP and anti-mouse HRP from Thermo Scientific.

Lehman S.L., Cerniglia G.J., Johannes G.J., Ye J., Ryeom S, & Koumenis C. (2015). Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress. PLoS Genetics, 11(6), e1005212.

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Quantitative Analysis of MYPT1 and RhoA Signaling

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NADP/NADPH Quantification Protocol

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Cells were lysed in NADP/NADPH extraction buffer (BioVision, Milpitas, CA, USA) for 10 min on ice. Protein concentrations were determined using the 660-nm protein assay reagent (Thermo Fisher). To detect the reduced form (NADPH) only, the lysates were incubated at 60 C for 30 min. Thereafter, NADP/NADPH-Glo assay (Promega) was used for NADPH determination. Sample absorbance or luminescence was recorded using the ARVO X5 multimode plate reader (PerkinElmer).

Toyomoto M., Inoue A., Iida K., Denawa M., Kii I., Ngako Kadji F.M., Kishi T., Im D., Shimamura T., Onogi H., Yoshida S., Iwata S., Aoki J., Hosoya T, & Hagiwara M. (2021). S1PR3-G₁₂-biased agonist ALESIA targets cancer metabolism and promotes glucose starvation. Cell chemical biology, 28(8).

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