By Christina M. Van Itallie, James M. Anderson (auth.), Kursad Turksen (eds.)
Since the preliminary discovery of the claudins, the sphere of tight junctions and mobile polarity has develop into more and more energized, often seeing that the claudins have supplied a chance to appreciate the molecular foundation of the tight junction functionality and their tissue particular roles. In Claudins: tools and Protocols, specialists and pioneers within the box offer an intensive number of innovations concerning those tight junction proteins. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, chapters contain short introductions to their respective issues, specific lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and key tips about troubleshooting and fending off recognized pitfalls.
Authoritative and functional, Claudins: tools and Protocols serves as an amazing advisor for all scientists looking to additional our knowing of this important sector of research.
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Extra info for Claudins: Methods and Protocols
Am J Physiol Cell Physiol 286, C1213–1228. S. (2008) Biology of claudins. Am J Physiol Renal Physiol 295, F867–876. , and Yang, Y. (2001) The roles of claudin superfamily proteins in paracellular transport. Traffic 2, 93–98. C. (2004) Barriers built on claudins. J Cell Sci 117, 2435–2447. Koval, M. (2006) Claudins: Key pieces in the tight junction puzzle. Cell Commun Adhes 13, 127–138. M. (2009) Physiology and function of the tight junction. Cold Spring Harbor Perspect Biol 1, a002584. G. (2003) Role of claudin interactions in airway tight junctional permeability.
Yu Abstract Claudins are tight junction membrane proteins that act as paracellular pores and barriers and regulate epithelial permeability to small ions. A key step in understanding the function of any claudin isoform is the in vitro measurement of its ion permeability and selectivity. Herein, we describe methods to generate clonal lines with stable inducible overexpression of claudins in Madin–Darby canine kidney epithelial cells, measure conductance and diffusion potentials in Ussing chambers, correct for liquid junction potentials, and derive quantitatively accurate values for individual ion permeabilities.
Once the Ringer saline is made, it is fairly stable and unused solution can be stored at 4°C (to prevent bacterial growth) for weeks to months. MDCK cell conductance and ion permeability are relatively insensitive to small changes in extracellular osmolality. , salts are fully dissociated in solution, the osmotic coefficient of mannitol is 1, and osmolality and osmolarity are equivalent). The commercial vector contains Moloney murine leukemia virus-based retroviral transcription and processing elements, together with a Tet-responsive promoter, and hygromycin resistance gene for selection in mammalian cells.