Coomassie plus protein reagent

Manufactured by Thermo Fisher Scientific

Coomassie Plus protein reagent is a colorimetric assay solution used to quantify total protein concentration in a sample. The reagent binds to proteins, resulting in a color change that can be measured spectrophotometrically.

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2 protocols using coomassie plus protein reagent

Western Blot Analysis of Phosphorylated Signaling Proteins

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For validation by Western blotting, four samples corresponding to four biological replicates from different cohorts of healthy donors were analysed per condition and experimental setting.
Proteins were precipitated from lysates in 20% trichloroacetic acid in acetone as previously described 15 , and protein pellets were resuspended in 50μl of sample buffer (65mM CHAPS, 5M urea, 2M thiourea, 0.15M NDSB-256, 30mM Tris, 1mM sodium vanadate, 0.1mM sodium fluoride, and 1mM benzamidine). Protein quantitation was done with Coomassie Plus protein reagent (Thermo Scientific) following manufacturer's protocol.
Protein separation by SDS-PAGE and subsequent immunoblotting were carried out as indicated in the Supplemental Methods. The following primary antibodies were used: rabbit anti-human p-PLCγ2 (Y759) (MAB7377, R&D systems) dilution 1/300; rabbit anti-p-Syk (Y525+Y526) (ab58575, Abcam) dilution 1/1000; rabbit anti-p-Src(Y418) (44660G, Fisher Scientific), dilution 1/1000; mouse anti-PLCγ2 (sc-5283, Santa Cruz Biotechnology) dilution 1/500; mouse anti-Syk (sc-1240, Santa Cruz Biotechnology) dilution 1/1000; rabbit anti-Src pan (44-656G,Invitrogen), dilution 1/1000. Further information on the statistical analysis can be found in the Supplemental Methods.

Izquierdo I., Barrachina M.N., Hermida-Nogueira L., Casas V., Morán L.A., Lacerenza S., Pinto-Llorente R., Eble J.A., de Los Ríos V., Domínguez E., Loza M.I., Casal J.I., Carrascal M., Abián J, & García A. (2020). A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade. Thrombosis and haemostasis, 120(2).

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Comprehensive Protein Analysis Protocol

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Cell lysates were prepared using 50 mM Tris, pH 8, 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 1% NP‐40 and 0.5% sodium deoxycholate lysis buffer containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Roche Applied Science, Indianapolis, Indiana). Protein concentration was determined by Bradford assay using Coomassie Plus Protein Reagent (Thermo Fisher Scientific). Proteins (50 μg) were resolved on a 4%‐12% Bis‐Tris gradient SDS‐PAGE (Bio‐Rad Laboratories) under reducing conditions and transferred to nitrocellulose membrane (Bio‐Rad Laboratories). Primary antibodies to p‐PI3K, ALDH1A1, nuclear factor κB (NF‐κB), p‐NF‐κB, SRC, p‐SRC, max, p27, p21, p‐c‐JUN, p53, p‐p53, Ku70, DNMT1, DNMT3a, DNMT3b, HDAC2, and β‐actin were purchased from Santa Cruz Biotechnology. All other antibodies used in this study were from Cell Signaling Technology. HRP‐conjugated secondary antibodies were from Santa Cruz Biotechnology. Protein bands were detected by SuperSignal West Dura Supstrate Western Blot Detection Reagent (Thermo Fisher Scientific).

Mrdenovic S., Zhang Y., Wang R., Yin L., Chu G.C., Yin L., Lewis M., Heffer M., Zhau H.E, & Chung L.W. (2019). Targeting Burkitt lymphoma with a tumor cell–specific heptamethine carbocyanine‐cisplatin conjugate. Cancer, 125(13), 2222-2232.

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