ZO Tight Junction Antibody Kit, BioAssay(TM)

Tight junctions are specialized regions of cell-cell contact that seal the paracellular space. TJ strands on adjacent cells are presumed to interact with each other to form a "molecular gasket" that prevents ions, water and other molecules from leaking between cells. In addition to this barrier function, the fence function of tight junctions plays an important role in maintaining epithelial cell polarity by blocking the diffusion of membrane proteins between apical (luminal) and basolateral cell surfaces. At one time, the only transmembrane protein known to be associated with tight junctions was occludin, a ~65kD protein with four transmembrane domains. Despite widespread expectation, a critical structural role for occludin in TJ strands was ruled out by the observation of apparently normal tight junctions formed between cells disrupted at both occludin alleles. Fortunately, a closer examination of isolated tight junctions uncovered two related ~22kD, four-transmembrane domain proteins, claudin-1 and claudin-2, with no similarity to occludin. In contrast to occludin, which induces only a small number of short strands at cell-cell contact sites when introduced into fibroblasts lacking tight junctions, claudin-1 and -2 induce networks of strands characteristic of true tight junctions.1,2 These findings suggest that\nclaudin-1 and -2 are major structural components of TJ strands and that occludin plays an accessory role. Claudins interact directly with the membrane-associated guanylate kinase homologues, ZO-1, ZO-2, and ZO-3, and are complexed with two other transmembrane proteins, occludin and junctional adhesion molecule.3 E-Cadherin is also important for the formation and maintenance of tight junctions.4 Cell-cell contacts containing E-Cadherin are crucial to the proper assembly and permanent sealing of tight junctions.5 To date, 20 claudins have been identified; studies suggest that the claudins comprising tight junctions are differentially expressed in different tissues. A number of human diseases are caused by claudin defects, which underscores the importance of claudins in human physiology. Deletion of the claudin-11 gene in mice results in disappearance of tight junction fibrils in testis and male sterility, as well as delayed axonal conduction rates in the CNS.7 An autosomal recessive form of deafness has been associated with human mutations in claudin-14.8 Claudin-7 expression is reportedly lower in invasive ductal breast carcinomas.\n\nKit Components:\nZ0775A: Ms/Mono; Z0775B: Rb/Poly; Z0775C: Ms/Mono; Z0775D: Rb/Poly; Z0775E: Rb/Poly; Z0775F: Ms/Mono.\nAll Mabs are IgG1, kappa and 20ug. The Pabs are IgG and 10ug.\n\nZ0775A, Z0775B: ZO-1; Z0775C, Z0775D: Occludin; Z0775E: Claudin-1; Z0775F: E-Cadherin\n\nZ0775A: Human, canine\nZ0775B: Human, mouse, rat, canine\nZ0775C: Human, rat, canine\nZ0775D: Human, canine\nZ0775E: Human, rat, canine\nZ0775F: Human\n\nApplications:\nZ0775A: Ms/Mono;Z0775C: Ms/Mono; Z0775F: Ms/Mono. \nSuitable for use in ELISA, Immunofluorescence and Western Blot\n\nZ0775B: Rb/Poly; Z0775D: Rb/Poly; Z0775E: Rb/Poly;Suitable for use in ELISA, Immunofluorescence, Immunoprecipitation, Western Blot and Immunohistochemistry. \n\nRecommended Dilution: \nELISA: 0.1-1ug/ml\nImmunofluorescence: 10-20ug/ml\nWestern blot: 0.5-3ug/ml\nImmunohistochemistry (with ZO-1 and Occludin pAbs and E-Cadherin mAb): 2-5ug/ml\nImmunoprecipitation (with ZO-1 and Occludin pAbs and E-Cadherin mAb): 2-5ug/ IP reaction.\nOptimal dilution determined by the researcher.\n\nPretreatment protocols for use with formalin-fixed, paraffin-embedded tissue sections.\n1. ZO-1 and Occludin pAbs: Protease (Sigma P-5147) at 1-2mg/ml for 10 minutes at 37 degrees C, after slides have been deparaffinized and rehydrated in ethanol. Normal human colon sections may be used as a positive control.\n2. E-Cadherin mAb: HIER with citrate buffer, pH 6.0 for 15 minutes at 95 degrees C, or boiling. Normal human colon sections may be used a positive control.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot and add glycerol (40-50%). Freeze at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

Z0775

Size

1Kit

Applications

ELISA, IF, IHC, IP, WB

Reactivities

Hum

Form

All antibodies are supplied as a liquid in PBS, pH 7.4, 0.1% sodium azide.

Purity

All antibodies are purified by affinity chromatography. The monoclonal antibodies are Protein A purified from mouse ascites and the polyclonal antibodies are antigen affinity purified.

Isotype

IgG1,k

References

1. Furuse, M., et al., J. Cell Biol. 143: 391-401 (1998). 2. Tsukita, S. and Furuse, M., Genes Cells 3: 569-573 (1998). 3. Keiskala, R.W., et al., Traffic 2(2): 93-98 (2001). 4. West, M.R., et al., Cell Commun. Adhes. 9(1): 29-44 (2002). 5. Contreras, R.G., et al., Pflugers Arch. 444(4): 467-475 (2002). 6. Morita, K., et al., Proc. Natl. Acad. Sci. USA 96: 511-516 (1999). 7. Gow, A., et al., Cell 99: 649-659 (1999). 8. Wilcox, E.R., et al., Cell 104: 165-172 (2001).

Additional Info

ZO-1, ZO-1, Occludin, Occludin, Claudin-1, E-Cadherin. Reactivity: Human, rat, canine, mouse.