By Gerard Drewes, Marcus Bantscheff
The multidisciplinary technology of chemical proteomics experiences how small molecules of man-made or typical beginning bind to proteins and modulate their functionality. In Chemical Proteomics: tools and Protocols, professional researchers within the box offer key suggestions to enquire chemical proteomics concentrating on analytical recommendations, how probes are generated, concepts for the invention of small molecule pursuits and the probing of aim functionality, and small molecule ligand and drug discovery. Written within the hugely profitable equipment in Molecular Biology™ sequence layout, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and key pointers on troubleshooting and warding off recognized pitfalls. Authoritative and functional, Chemical Proteomics : tools and Protocols seeks to supply methodologies that may give a contribution to a much broader program of chemical proteomics equipment in biochemical and cellphone organic laboratories.
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Extra info for Chemical Proteomics: Methods and Protocols (Methods in Molecular Biology, v803)
Transfer the gel into a clean Petri dish after the second wash step to keep the background as low as possible. 6. 05% formaldehyde. Rock the Petri dish until the solution changes to a yellow color. Remove the liquid and allow the development without liquid. If the staining is not sufficient, repeat the developing step. 7. When all the bands are visible on the gel, quench the developer by removing the liquid from the Petri dish and adding 5% acetic acid. Leave the gel in the acetic acid for a minimum of 5 min.
J. Virol. 83, 10058–10074. Chemical Proteomics in Drug Discovery 21 28. Gharbi, S. , Zvelebil, M. , Shuttleworth, S. , Timms, J. , and Waterfield, M. D. (2007) Exploring the specificity of the PI3K family inhibitor LY294002. Biochem. J. 404, 15–21. 29. , and Heck, A. J. (2010) Target profiling of a small library of phosphodiesterase 5 (PDE5) inhibitors using chemical proteomics. ChemMedChem. 5, 1927–1936. 30. Patricelli, M. , Szardenings, A. , Nomanbhoy, T. , and Kozarich, J. W. (2007) Functional interrogation of the kinome using nucleotide acyl phosphates.
Covalent active-site labeling probes are typically biotin conjugates and have been applied to kinases (30), GTPases (31), methylases (32), dehydrogenases (33), serine-, cysteine-, metallo-, and proteasomal proteases (23, 34, 35), and HDACs (36). These methodologies typically generate protein affinity profiles for the immobilized compounds, which may reveal novel target candidates, but precautions must be taken to avoid false positives due to the background problems caused by nonspecific interactions with abundant proteins.