Cell Signaling Technology

Product Pathways - Adhesion

Tight Junction Antibody Sampler Kit #8683

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
CD2AP Antibody #2135 40 µl W H M R Mk 80 Rabbit
Claudin-1 Antibody #4933 40 µl W IP H (M) (R) 20 Rabbit
ZO-1 (D7D12) Rabbit mAb #8193 40 µl W IP H Mk 220 Rabbit IgG
ZO-2 Antibody #2847 40 µl W IF-IC H M R Mk B Dg 150 Rabbit
ZO-3 (D57G7) XP® Rabbit mAb #3704 40 µl W IF-IC H 140 Rabbit IgG
Afadin Antibody #6492 40 µl W H M R Mk 205 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  B=Bovine  Dg=Dog
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

CD2AP Antibody recognizes endogenous levels of total CD2AP protein. Claudin-1 Antibody recognizes endogenous levels of total claudin-1 protein. Based on sequence similarity, Claudin-1 Antibody may cross-react with claudin-2 protein. ZO-1 (D7D12) Rabbit mAb recognizes endogenous levels of total ZO-1 protein. ZO-2 Antibody recognizes endogenous levels of total ZO-2 protein. ZO-3 (D57G7) XP® Rabbit mAb detects endogenous levels of total ZO-3 protein. Afadin Antibody recognizes endogenous levels of total afadin protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from K562, A431, and COS cells using CD2AP Antibody #2135.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using ZO-2 Antibody #2847.

Western Blotting

Western Blotting

Western blot analysis of extracts from MCF-7, HT-29, and HCT-15 cells using ZO-3 (D57G7) XP® Rabbit mAb #3704.


Western Blotting

Western Blotting

Western blot analysis of extracts from A-431 and ZR-75 cells using Claudin-1 Antibody #4933.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Afadin Antibody #6492.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using ZO-1 (D7D12) Rabbit mAb #8193.


Description

The Tight Junction Antibody Sampler Kit provides an economical means to evaluate the presence of a number of proteins involved in tight junctions. The kit contains enough primary antibodies to perform four western blot experiments per primary antibody.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues near the carboxy terminus of human CD2AP protein, surrounding Pro574 of human afadin, mouse claudin-1 protein, or mouse ZO-2 protein. Antibodies are purified using protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with synthetic peptides near the carboxy terminus of human ZO-1 protein or human ZO-3 protein.

Background

Tight junctions, or zona occludens, form a continuous barrier to fluids across the epithelium and endothelium. They function in regulation of paracellular permeability and in the maintenance of cell polarity, blocking the movement of transmembrane proteins between the apical and basolateral cell surfaces (reviewed in 1). Tight junctions are composed of claudin and occludin transmembrane proteins, which join the junctions to the cytoskeleton (1,2). The claudin family is composed of 23 integral membrane proteins, and their expression, which varies among tissue types, may determine both the strength and properties of the epithelial barrier (2,3). Zona occludens proteins ZO-1, -2, and -3 (also known as TJP1, 2, and 3) are peripheral membrane adaptor proteins that link junctional transmembrane proteins such as occludin and claudin to the actin cytoskeleton (reviewed in 4). ZO-1 and ZO-2 are required for tight junction formation and function (5,6). In subconfluent proliferating cells, ZO-1 and ZO-2 have been shown to colocalize to the nucleus and play a role in transcriptional regulation (7-9). Exogenous expression of the amino terminal portion of ZO-3 exerts a dominant negative effect that interferes with assembly of tight junctions and adherens junctions (10). ZO-1 has been shown to interact with afadin prior to the formation of tight junctions (11). Recent work has also shown that afadin is involved in controlling the directionality of cell movement when it is localized at the leading edge of moving cells (12,13). CD2AP is a scaffolding protein that is thought to link membrane proteins to the cytoskeleton (14-16). It plays a role in the formation of tight junctions in specialized cell types such as the slit diaphragm of the kidney glomerulus (17). CD2AP is also involved in the immunological synapse between CD2-expressing T cells and antigen presenting cells (18). Research studies have shown that interaction between CD2AP and other cytoskeletal proteins may regulate the endocytosis of EGFR (16).

  1. Shin, K. et al. (2006) Annu Rev Cell Dev Biol 22, 207-35.
  2. Oliveira, S.S. and Morgado-Díaz, J.A. (2007) Cell Mol Life Sci 64, 17-28.
  3. Hewitt, K.J. et al. (2006) BMC Cancer 6, 186.
  4. Matter, K. and Balda, M.S. (2007) J Cell Sci 120, 1505-11.
  5. Hernandez, S. et al. (2007) Exp Cell Res 313, 1533-47.
  6. Umeda, K. et al. (2006) Cell 126, 741-54.
  7. Betanzos, A. et al. (2004) Exp Cell Res 292, 51-66.
  8. Traweger, A. et al. (2003) J Biol Chem 278, 2692-700.
  9. Huerta, M. et al. (2007) Mol Biol Cell 18, 4826-36.
  10. Wittchen, E.S. et al. (2000) J Cell Biol 151, 825-36.
  11. Ooshio, T. et al. (2010) J Biol Chem 285, 5003-12.
  12. Miyata, M. et al. (2009) J Cell Sci 122, 4319-29.
  13. Miyata, M. et al. (2009) J Biol Chem 284, 24595-609.
  14. Kirsch, K.H. et al. (1999) Proc Natl Acad Sci U S A 96, 6211-6.
  15. Kirsch, K.H. et al. (2001) J Biol Chem 276, 4957-63.
  16. Lynch, D.K. et al. (2003) J Biol Chem 278, 21805-13.
  17. Kawachi, H. et al. (2006) Nephrology (Carlton) 11, 274-81.
  18. Hutchings, N.J. et al. (2003) J Biol Chem 278, 22396-403.

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