Mammary Gland Protein Extraction and Quantification

Total protein was extracted from mammary glands by using the radioimmunoprecipitation assay buffer (R2078, Sigma, St Louis, MO, USA) with protease and phosphatase inhibitors (33 (link), 34 (link)). Protein concentrations of mammary extracts were quantified by using the bicinchoninic acid protein assay (Thermo Scientific). Sandwich enzyme-linked immunosorbent assays were performed to quantify estradiol (Biovision, Milpitas, CA, USA), progesterone (MyBioSource, San Diego, CA, USA), estrogen receptor 1 and 2, and progesterone receptor (LifeSpan BioSciences, Seattle, WA, USA) in mammary glands. Samples were read within the linear range of the assay. The accuracy of the analysis was confirmed by the controls provided in each kit.

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Sundaram S., Johnson L.K, & Yan L. (2020). High-Fat Diet Alters Circadian Rhythms in Mammary Glands of Pubertal Mice. Frontiers in Endocrinology, 11, 349.

Publication 2020

radioimmunoprecipitation assay buffer bicinchoninic acid protein assay estradiol progesterone

Assay Bicinchoninic acid Buffer Enzyme linked immunosorbent assays Estradiol Estrogen receptor 1 Inhibitors Mammary glands Phosphatase Progesterone Progesterone receptor Protease Protein Radioimmunoprecipitation assay

Corresponding Organization : Agricultural Research Service

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Variable analysis

independent variables

Protein extraction using radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors

dependent variables

Total protein concentration in mammary glands
Estradiol levels in mammary glands
Progesterone levels in mammary glands
Estrogen receptor 1 and 2 levels in mammary glands
Progesterone receptor levels in mammary glands

control variables

Samples were read within the linear range of the assay
The accuracy of the analysis was confirmed by the controls provided in each kit

controls

Positive controls: Controls provided in each kit to confirm the accuracy of the analysis
Negative controls: Not explicitly mentioned

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