By Kathleen M. Eyster
This volume examines a wide range of significant applied sciences for advancing our figuring out of the receptor-mediated activities of estrogen. The protocols during this e-book variety from general tools and important laboratory workhorses, similar to receptor binding assays and western blot, to more moderen applied sciences akin to RNAseq and proximity ligation assay. Chapters additionally speak about protocols from a wide variety of tissue forms to illustrate the range of estrogen receptor results. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, easily reproducible laboratory protocols, and pointers on troubleshooting and keeping off recognized pitfalls.
Cutting-edge and thorough, Estrogen Receptors: equipment and Protocols is an in depth and useful source for scientists who're intrigued through the various elements of estrogen.
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Extra resources for Estrogen Receptors: Methods and Protocols
Example text
6. Scintillation fluid (Scintiverse®). 7. Scintillation counting vials. 3 Methods Following are two specific methods, one for radioligand binding on total membrane extracts [8], and the other for assessing the binding of a given ligand (for our purposes we have used [3H] 2ME2) for ER membrane localized GPR30 receptors [9]. All steps of membrane isolation must be performed at 4 °C or on ice, while radioligand binding may proceed at room temperature (22 °C). 1 Total Cell Membrane Preparation 1. Grow cells (rat liver epithelial cells [3]) to 90 % confluence in 100 mm cell culture plates under conditions required by the experiment.
05) [6, 19]. 3. Firstly upload the ChIP-seq data as shown in Fig. 1. 4. Next use “Generate Tracks > Generate segment track from gene IDs” to obtain genomic intervals from the ENSEMBL gene IDs of DEGs. These should be uploaded into the tool as a series of comma-separated values as shown by the demo data. If necessary genomic intervals can be lifted from one genome build to another by the “Lift-Over > Convert genomic coordinates” tool. 5. Use “Operate on Genomic Intervals > Get flanks” to extend gene regions by a pre-specified number of bases in each direction (Fig.
The probes are typically designed to span exon-exon junctions so as to decrease the likelihood of amplification from contaminating genomic DNA (see Note 1). The oligomer probes carry a fluorescent reporter dye at one end and a quencher dye at the other end. As long as the reporter dye and the quencher are held in close proximity by their attachment to the oligomer probe, no fluorescence is released from the reporter dye. The release of the fluorophore from the oligomer probe relies on the 5′ nuclease activity of the DNA polymerase [9].