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 mobilephone polarity has turn into more and more energized, in general 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: equipment and Protocols, specialists and pioneers within the box supply an in depth selection of options concerning those tight junction proteins. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, chapters comprise short introductions to their respective themes, certain lists of the required fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key pointers on troubleshooting and fending off recognized pitfalls.
Authoritative and functional, Claudins: equipment and Protocols serves as an excellent advisor for all scientists trying to extra our knowing of this very important zone of research.
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Extra info for Claudins: Methods and Protocols
5 × 105 cells total/100-mm dish or 3 × 105 total cells/35-mm dish for coimmunopurification or immunofluorescence, respectively. Cells will be ready for analysis 3 days after plating (see Note 11). 2. Cell cocultures on 25-mm glass coverslips in 35-mm dishes should be nearly confluent prior to processing for immunofluorescence. Include monocultures as controls. Use PBS + Ca + Mg when processing cells for immunofluore scence. Wash cell cocultures 3 × 1 ml PBS at room temperature. Remove the PBS and replace with 1 ml 1:1 methanol/acetone (made fresh).
Periodically check stably transfected HeLa cell clones by immunofluorescence to insure that the cells have retained claudin expression. If the cocultures do not show an adequate number of heterotypic interfaces, they can be propagated and re-trypsinized prior to use for experiments. Also, claudin expression can have a differential effect of the growth of cell clones. For instance, we have found that HeLa/claudin-3 cells tend to be slower growing than other HeLa/claudin transfectants. If so, then compensate for this by altering the ratio of cells initially plated in the cocultures.
Ward and M. Activate a piece of PVDF transfer membrane cut to match the gel in 100% methanol for 2 min, rinse with H2O, then incubate in transfer buffer for ~30 min. Pre-soak two pieces of pre-cut blotting paper for each gel in transfer buffer. Assemble the one piece of blotting paper, PVDF membrane, gel and then another piece of blotting paper in the semi-dry transfer apparatus. Run for 1 h at 24 V constant voltage. After the transfer is complete, place the PVDF membrane in 20 ml of Blotto and incubate at either room temperature for 1 h or overnight at 4°C.
Claudins: Methods and Protocols by Christina M. Van Itallie, James M. Anderson (auth.), Kursad Turksen (eds.)