Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin
The estrogen receptor α ligand-binding domain (ERα-LBD) binds the natural hormone 17β-estradiol (E2) to induce transcription and cell proliferation. This process occurs with the contribution of protein and peptide partners (also called coactivators) that can modulate the structure of ERα, and therefore its specificity of action. As with most transcription factors, ERα exhibits a high content of α helix, making it difficult to routinely run spectroscopic studies capable of deciphering the secondary structure of the different partners under binding conditions. Ca2+-calmodulin, a protein als...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Analysis of Interaction of Estradiol with Estrogen Receptor by NMR Spectroscopy
Following binding to estradiol, estrogen receptors (ER) α and ERβ recruit a number of interacting proteins and mediate a plethora of functions. The binding of estrogen with the receptors shows changes in the resonance structure and movement of protons. We cloned ERβ and its trans-activation domain (TAD) and ligand-binding domain (LBD), expressed them in prokaryotic expression vectors, purified them, and studied their interaction with estradiol. In this chapter, a detailed method of preparation of recombinant proteins, SDS-PAGE, silver staining, and NMR are described. Such methods are useful to check the bio...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Gold Nanoparticle-Based Förster Resonance Energy Transfer (FRET) Analysis of Estrogen Receptor: DNA Interaction
Estrogen receptors play critical roles in regulating genes responsible for development and maintenance of reproductive tissues and other physiological function. The interaction of ERs with DNA sequences, known as estrogen response elements (EREs) (a palindromic repeat separated by three-base spacer, 5′GGTCAnnnTGACC-3′), is required for estrogen regulation of target gene expression. Here, we describe a simple “mix-and-measure”-based method for detecting ER:ERE interactions using ERE-immobilized metal nanoparticles and water-soluble conjugated polyelectrolytes (CPEs) as cooperative sensing elements. T...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Purification of Histone Lysine Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from Estrogen Receptor α
Methylation of estrogen receptor α by the histone lysine methyltransferase SMYD2 regulates ERα chromatin recruitment and its target gene expression. This protocol describes SMYD2 purification and crystallization of SMYD2 in complex with an ERα peptide. Recombinant SMYD2 is overexpressed in Escherichia coli cells. After release from the cells by French Press, SMYD2 is purified to apparent homogeneity with multiple chromatography methods. Nickel affinity column purifies SMYD2 based on specific interaction of its 6×His tag with the bead-immobilized nickel ions. Desalting column is used for protein buff...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
In Situ Hybridization of Estrogen Receptors α and β and GPER in the Human Testis
In situ hybridization (ISH) is an excellent method for detecting RNA in histological sections, both to detect gene expression and to assign gene expression to a distinct cell population. Therefore, ISH may be used in basic cell biology to detect the expression of certain genes within a tissue containing various cell populations. Here, we describe the detection and cellular localization of three estrogen receptors, both isoforms of the genomic estrogen receptor (ERα and ERβ) as well as the membrane-bound G-protein-coupled estrogen receptor 1 (GPER) in the human testis. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Live-Cell Imaging of the Estrogen Receptor by Total Internal Reflection Fluorescence Microscopy
Trafficking studies of plasma membrane-localized intracellular estrogen receptors have mainly relied on biochemical and histological techniques to locate the receptor before and after estradiol stimulation. More often than not these experiments were performed using postmortem, lysed, or fixed tissue samples, whose tissue or cellular structure is typically severely altered or at times completely lost, making the definitive localization of estrogen receptors difficult to ascertain. To overcome this limitation we began using total internal reflection fluorescence microscopy (TIRFM) to study the trafficking of plasma membrane ...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Colocalization of Estrogen Receptors with the Fluorescent Tamoxifen Derivative, FLTX1, Analyzed by Confocal Microscopy
Tamoxifen is a selective estrogen receptor modulator that competitively binds the ligand-binding domain of estrogen receptors. Binding of tamoxifen displaces its cognate ligand, 17β-estradiol, thereby hampering the activation of estrogen receptors. Cellular labeling of ER is typically carried out using specific antibodies which require permeabilization of cells, incubation with secondary antibodies, and are expensive and time consuming. In this article, we describe the usefulness of FLTX1, a novel fluorescent tamoxifen derivative, which allows the labeling of estrogen receptors in immunocytochemistry and immunohistoch...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Assessment of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate Endometrium, Placenta, and Fetal Adrenal in Response to Estrogen
We describe herein the PLA method and its applicability to quantify the effects of estrogen on expression of angioregulatory factors, e.g., angiopoietin-1 (Ang-1) in the endometrium, vascular endothelial growth factor (VEGF) in the placenta, and melanocortin 2 receptor (MC2R)/accessory protein (MRAP) in the fetal adrenal of the nonhuman primate. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Shotgun Proteomics Analysis of Estrogen Effects in the Uterus Using Two-Dimensional Liquid Chromatography and Tandem Mass Spectrometry
Shotgun (gel-free) proteomics is a useful approach to perform identification and relative quantification of protein in complex mixtures such as tissue homogenates, biological fluids, cell lysates, and extracellular proteins. Incorporation of separative and analytical techniques such as two-dimensional liquid chromatography at nanoscale (2D-nanoLC) coupled to tandem mass spectrometry (MS/MS analysis) into the shotgun protocol provides an excellent strategy. This chapter describes the application of the shotgun proteomics protocol to evaluate the identity and expression analysis of proteins from rat uterus after estrogen (et...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
DNA Microarray Analysis of Estrogen-Responsive Genes
DNA microarray is a powerful, non-biased discovery technology that allows the analysis of the expression of thousands of genes at a time. The technology can be used for the identification of differential gene expression, genetic mutations associated with diseases, DNA methylation, single-nucleotide polymorphisms, and microRNA expression, to name a few. This chapter describes microarray technology for the analysis of differential gene expression in response to estrogen treatment. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Detection of Endogenous Selective Estrogen Receptor Modulators such as 27-Hydroxycholesterol
The estrogen receptors (ERs) belong to the nuclear receptor superfamily, and as such act as ligand inducible transcription factors, mediating the effects of estrogens. However, their pharmacology is complex, having the ability to be differentially activated by ligands. Such ligands possess the ability to behave as either ER-agonists or ER-antagonists, depending on the cellular and tissue context, and have been termed Selective Estrogen Receptor Modulators (SERMs). Several SERMs have been identified with clinical relevance such as tamoxifen and raloxifene. Recently, 27-hydroxycholesterol has been characterized as the first ...
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
Estrogen Receptor- & alpha; Knockout Mice
Tissue specific knockout mice are valuable tools to study gene function in vivo. The method uses the Cre/loxP system in which loxP sites are cloned into the genome surrounding one or more exons of a gene and the targeted exon(s) are deleted when the Cre enzyme is expressed. Mouse lines that are prepared for the generation of knockout ERα mice have been developed independently by many research groups and the number of available transgenic mouse lines that express Cre under tissue specific promoters is large. Here, we describe how tissue specific ERα knockout mice are generated. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
Detection and Functional Analysis of Estrogen Receptor & alpha; Phosphorylated at Serine 216 in Mouse Neutrophils
Serine 216 constitutes a protein kinase C phosphorylation motif located within the DNA binding domain of estrogen receptor & alpha; (ER & alpha;). In this chapter we present experimental procedures confirming that mouse ER & alpha; is phosphorylated at serine 216 in peripheral blood neutrophils and in neutrophils that infiltrate the uterus, as well as the role of phosphoserine 216 in neutrophil migration. A phospho-peptide antibody ( & alpha;P-S216) was utilized in Western blot, immunohistochemistry, and double immunofluorescence staining to detect this phosphorylation of an endogenous ER & alpha;. Both immunohistochemistr...
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
Silencing Estrogen Receptor- & beta; with siRNA in Cultured Cells
Estrogen receptors & alpha; and & beta; (ER & alpha; and ER & beta;) are the two genomic estrogen receptors. ER & beta; was the second of these receptors to be discovered; its structure is similar to that of ER & alpha; but they are different in histological distribution. However, the functions of ER & alpha; versus ER & beta; are still unclear. The ability of small interfering RNAs (siRNAs) to silence gene expression has proven to be invaluable for studying gene function in cultured mammalian cells. This chapter describes the use of siRNA to inhibit the expression of ER & beta; in renal cell carcinoma (RCC) and to further...
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
Silencing Estrogen Receptor- & alpha; with siRNA in the Intact Rodent Brain
The ability to silence the expression of gene products in a chemically, spatially, and temporally specific manner in the brains of animals has enabled key breakthroughs in the field of behavioral neuroscience. Using this technique, estrogen receptor alpha (ER & alpha;) has been specifically implicated in a multitude of behaviors in mice, including sexual, aggressive, locomotor, and maternal behaviors. ER & alpha; has been identified in a variety of brain regions, including the medial preoptic area, ventromedial hypothalamus, and amygdala. In this chapter we describe the techniques involved in the generation of the small ha...
Source: Springer protocols feed by Biochemistry - October 11, 2015 Category: Biochemistry Source Type: news
