By Kris De Boeck, Melissa Ashlock (auth.), Margarida D. Amaral, Karl Kunzelmann (eds.)
Despite the various milestones in cystic fibrosis (CF) examine, growth in the direction of curing the sickness has been sluggish, and it truly is more and more tough to know and use the already large and nonetheless becoming variety of various tools at present hired to review CF so that it will realize it in its multidisciplinary nature. Cystic Fibrosis: prognosis and Protocols goals to supply the CF examine neighborhood and similar researchers with a truly wide selection of fine quality experimental instruments, as a great way to know and use classical and novel equipment utilized to cystic fibrosis. Volume I: techniques to check and proper CFTR Defects specializes in the cystic fibrosis transmembrane conductance regulator (CFTR) and its expression, biogenesis, constitution, and serve as by way of the defects inflicting CF. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and pointers on troubleshooting and averting recognized pitfalls.
Comprehensive and useful, Cystic Fibrosis: prognosis and Protocols will supply readers with optimum operating instruments to deal with urgent questions within the most sensible technical method, whereas supporting we all, as a study and medical group, to maneuver swifter hand-in-hand towards unravelling the secrets and techniques of this tough affliction and medication it.
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Extra info for Cystic Fibrosis: Diagnosis and Protocols, Volume I: Approaches to Study and Correct CFTR Defects
2006) Rescue of F508 CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules. Am. J. Physiol. Lung Cell Mol. Physiol. 290, L1117–L1130. 6. , Lukacs, G. , Galietta, L. , et al. (2005) Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J. Clin. Invest. 115, 2564–2571. 7. Carlile, G. , Teske, K. , Hanrahan, J. W. et al. (2007) Correctors of protein trafficking defects identified by a novel high-throughput screening assay.
A poor transfection (low expression) will also affect the intensity of signal obtained. 15. An alternative method for labeling cell surface proteins is to use NHS–biotin (Pierce) that labels amino groups. 16. If HEK 293 cells detach too easily, then transfect COS-1 cells. 17. Cross-linking causes CFTR to migrate slower on SDSPAGE gels. 18. The pcDNA3 vector already contains a neomycin resistance gene, so co-transfection is not necessary. Some of our CFTR cDNAs have been cloned into the pMT21 vector that does not contain a neomycin resistance gene, so co-transfection must be performed with PWL-neo.
For BHK cells, the vector and media are the same as described for HEK 293 cells. BHK cells are co-transfected with the CFTR plasmid and vector containing the neomycin resistance gene (such as pWL-neo) at a 1:9 ratio (see Note 18). The next day, media is changed to plain media. The following day, media is added that contains 1 mg/ml G418. Colonies are selected after 7–10 days and CFTR-expressing clones identified and expanded as described above. 2. For iodide efflux measurements, stable lines expressing wildtype CFTR, F508del-CFTR, or control cells that do not express CFTR are used.
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