Validation of specificity and enrichment of tight junction complexes. (A) Silver stained gel showing steps of purification. Lane 1, whole cell lysate; lane 2, 30,000 × g supernatant; lane 3, 100,000 × g membrane; Lane 3', output 100,000 × g membrane after immuno-isolation, Lane 3", output 100,000 × g membrane after control immuno-isolation; lane 4, immuno-isolated tight junction complexes; lane 5, control immuno-isolation. MW markers are 200, 116, 96, 66, 45, 38, 25, 14. (b) Western blots of tight junction markers occludin and claudin-1. Lanes 1–5, same as A. (C) Negative staining of complexes showing assemblies of a heterogeneous population of proteins in various sizes. Control immunopurification shows relatively clean background. Scale bars, 100 nm. (D) Higher magnification of tight junction complexes shows globular proteins linked together forming beaded-necklace arrays reminiscent of tight junction seen in vivo (see Results).