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 telephone polarity has develop into more and more energized, generally since 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 offer an in depth selection of concepts concerning those tight junction proteins. Written within the hugely winning Methods in Molecular Biology™ sequence layout, chapters comprise short introductions to their respective subject matters, designated lists of the mandatory fabrics and reagents, step by step, with ease reproducible laboratory protocols, and key tips about troubleshooting and warding off recognized pitfalls.
Authoritative and useful, Claudins: equipment and Protocols serves as an excellent advisor for all scientists trying to additional our knowing of this important zone of research.
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Extra info for Claudins: Methods and Protocols
Scrape the cells off the plates into the Lysis Buffer and transfer to a 15-ml conical polyethylene tissue culture tube. Centrifuge at 500 ´ g for 5 min at 4°C. 1% Triton X-100. 5ml Eppendorf tube (see Note 13). Incubate on ice for 10 min. Sonicate while on ice with a narrow tip XL-2000 probe Misonix sonicator. Use 3 × 3 s pulses at 80% to shear the nuclear material. Microfuge at 16,000 × g for 10 min. 5-ml Eppendorf tube containing 50 ml of 2× sample buffer. This is the total lysate sample.
Thoroughly mix by flicking with your finger, do not vortex. Incubate at 4°C for 30 min with intermittent mixing. Prepare separate batches using a different rabbit antibody for each claudin to be analyzed. DynaMag™-2 magnetic isolation rack (Invitrogen). 5 mg bromophenol blue. This is stable 4°C or room temperature (w/o DTT) for a few months. Add 100 mg dithiothreitol (DTT) to 1 ml 2× SB to make working stocks, unused portions can be stored at −20°C up to 1 month. 4. Ammonium persulfate (APS), 10% solution in H2O, store at 4°C up to 1 month.
Also, using mouse monoclonal antibodies for immunoblots from samples where rabbit IgG was used for coimmunopurification reduces detection of IgG band. Since the IgG light chain migrates close to the position where claudins migrate (~22 kDa), this cross-species approach improves the interpretability of coimmunopurification immunoblots. We have found that hand cast SDS–PAGE gels provide more consistent analysis for claudins than pre-cast gels. HeLa cell lines show significant clonal variation; several lines have a more fibroblastic morphology and are less suitable for 24 C.