Gene Regulation: Methods and Protocols (Methods in Molecular Biology)
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In this volume of Methods in Molecular Biology™, expert investigators offer comprehensive, complementary, and cutting-edge technologies for studies of gene regulation. The chapters of Gene Regulation: Methods and Protocols are organized to provide an integrated and a coherent view of control systems and their associated components. The protocols are broad in their scope. They include molecular, biochemical, spectroscopic techniques as well as high throughput strategies. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Comprehensive and broad in their scope, the protocols are useful to researchers in many disciplines including molecular biology, genomics, biochemistry, biomedicine, nutrition, and agricultural sciences.
insulators, and locus control regions (2), and thus encompass genomic sequences of substantial biological importance. Recent advances Minou Bina (ed.), Gene Regulation: Methods and Protocols, Methods in Molecular Biology, vol. 977, DOI 10.1007/978-1-62703-284-1_2, © Springer Science+Business Media, LLC 2013 13 14 G. Ling and D.J. Waxman TF binding sites exposed Epigenetic changes Gene expression Heterochromatin DNase I Euchromatin Fig. 1. DNase hypersensitivity identifies open genomic
Biochem 188:245–254 10. Bronstein I, Fortin J, Stanley PE et al (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal Biochem 219: 169–181 11. Ow DW, de Wet JR, Helinski DR et al (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234:856–859 12. de Wet JR, Wood KV, Deluca M et al (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737 13. Fraga H, Fernandes D, Fontes R et
range, increase or decrease the output on the sonicator and the number of pulses or cycles during sonication. 6. After you have optimized the sonication conditions, determine the efficiency of HaloTag binding (Subheading 3.7) to ensure that the sonication conditions are optimal for HaloLink Resin capture. 3.7. Analysis of HaloTag Fusion Protein Binding Efficiency to HaloLink Resin This protocol requires the use of a fluorimager for detection of fluorescent bands within an SDS gel. If such
DNA. To obtain higher amounts of DNA, increase the number of HaloCHIP reactions performed, following the protocol as recommended, and pool purified DNA at the end of the process. This DNA can then be lyophilized to the desired concentration. 6. HaloLink resin will settle quite quickly in ethanol, therefore it is important to mix gently right before pipetting to obtain a uniform solution. 7. Excessive cross-linking can reduce the ability of the HaloTag protein to bind to its resin. If this is
determination of DNA concentration in small volumes. 6. Gel drier for drying short and long polyacrylamide gel. 3. Methods We have developed a strategy for the detection of protein binding regions in long (<10 kb) fragments of genomic DNA (Fig. 1) (12). With this strategy restriction fragments, containing transcription factor binding sites, derived from promoter region of any gene may be identified by combination of EMSA and denaturing PAGE. The following is a detailed description of the methods