By Kathleen M. Eyster
This volume examines a wide range of important applied sciences for advancing our knowing of the receptor-mediated activities of estrogen. The protocols during this publication diversity from regular tools and very important laboratory workhorses, resembling receptor binding assays and western blot, to more moderen applied sciences corresponding to RNAseq and proximity ligation assay. Chapters additionally talk about protocols from a large variety of tissue varieties to illustrate the range of estrogen receptor results. Written within the hugely profitable Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and pointers on troubleshooting and warding off identified pitfalls.
Cutting-edge and thorough, Estrogen Receptors: equipment and Protocols is an in depth and worthy source for scientists who're intrigued via the various aspects of estrogen.
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Extra info for Estrogen Receptors: Methods and Protocols
Transfer the supernatant to a fresh tube and centrifuge at 45,000 × g for 20 min. 7. 4 and centrifuge at 45,000 × g for 20 min. 8. Suspend the resulting pellet in binding buffer without BSA and use immediately for radioligand binding study or store in aliquots at −80 °C (see Note 4). 9. Determine the protein concentration before the binding assay (We use the Bio-Rad protein assay based on the Bradford method). 14 Thomas Thekkumkara et al. 2 Preparation of Endoplasmic Reticulum Membranes 1. Grow cells (rat liver epithelial cells ) to 90 % confluence in a 75 cm2 cell culture flask under conditions required by the experiment.
12. When the sample of RNA is limited or when it is necessary to maintain long-term storage of a sample of cDNA, it may be preferable to perform a two-step reaction in which the reverse transcription reaction is carried out in a conventional PCR machine, and then the real time PCR reaction is performed using cDNA from the first step. , QIAquick PCR Purification Kit, Qiagen) as the residual nucleotides, salts, and buffers from the RT reaction may interfere with the subsequent real-time PCR reaction.
3. , Internet Explorer, Mozilla Firefox or Google Chrome). 4. A high-speed Internet connection. 1 The Genomic HyperBrowser 1. no/hb/). 2. Prepare ChIP-seq and transciptomic datasets for upload. ChIP-seq files should be a set of tab-delimited genomic coordinates corresponding to each peak in the format: Chromosome Start Stop Transcriptomic files should be a list of differentially expressed genes (DEGs) as ENSEMBL IDs (see Note 1 for methods for converting between different forms of gene ID). 05) [6, 19].
Estrogen Receptors: Methods and Protocols by Kathleen M. Eyster