By Xiongjie Jin, Binnur Eroglu, Demetrius Moskophidis (auth.), Stuart K. Calderwood, Thomas L. Prince (eds.)
The proteome comprises a fancy mix of proteins every one of which must be folded thoroughly with the intention to functionality for the well-being of the organism, and lots of of those proteins require molecular chaperones to arrive the proper conformation and, every now and then, to stay in a folded shape. In Molecular Chaperones: equipment and Protocols, professional researchers tackle a large choice of methods to the research those mechanisms, that includes the workings of warmth surprise proteins and warmth surprise transcription components, in vitro and in vivo. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, chapters positive factors introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Authoritative and state-of-the-art, Molecular Chaperones: equipment and Protocols serves as an incredible consultant for all scientists who desire to pursue this very important organic motion and its influence on human future health and disease.
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Extra resources for Molecular Chaperones: Methods and Protocols
Luciferase Reporter Assays for HSF Activity To construct an intracellular reporter of HSF activity (pGL. Basic plasmid (Promega). The HSP70B gene is almost entirely silent at physiological temperatures, but powerfully activated by heat shock (34). Basic. Basic. 1 (−) expression vector (Invitrogen) at the XhoI and EcoRI sites (36). 1(+) vector at the XhoI and EcoRI sites to produce pHSF2A (37). To assay of HSF1 transcriptional activity in HeLa cells, the cells are maintained in HAM’s F-12 (Mediatech) with 10% heatinactivated fetal bovine serum (FBS).
Wang G, Ying Z, Jin X, Tu N, Zhang Y, Phillips M, Moskophidis D, Mivechi NF. Essentail requirement for both hsf1 and hsf2 transcriptional activity activity in spermatogenesis and male fertility. Genesis 2004;38:66–80. 29. Bu L, Jin Y, Shi Y, et al. Mutant DNA-binding domain of HSF4 is associated with autosomal dominant lamellar and Marner cataract. Nat Genet 2002;31:276–8. 30. Koni PA, Joshi SK, Temann UA, Olson D, Burkly L, and Flavell RA. Conditional vascular cell adhesion molecule 1 deletion in mice: Impaired lymphocyte migration to bone marrow.
Measuring HSF1 Binding to HSP Promoters In Vivo by the Chromatin Immunoprecipitation Assay ChIP offers an attractive solution to transcription analysis by combining the specificity of immunoprecipitation and the sensitivity of PCR (see Note 2). The method allows monitoring of the interactions between DNA and transcription factors and/or components of chromatinremodeling complexes, but it is technically challenging due to the low abundance and/or only temporary interactions of these proteins (31).
Molecular Chaperones: Methods and Protocols by Xiongjie Jin, Binnur Eroglu, Demetrius Moskophidis (auth.), Stuart K. Calderwood, Thomas L. Prince (eds.)