Western blot analysis of extracts from NTERA-2 cl.D1 and DU 145 cells using Claudin-6 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, DU 145 is low or negative for expression of Claudin-6.
Immunoprecipitation of Claudin-6 from OVCAR3 extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is Claudin-6 Antibody. Western blot analysis was performed using Claudin-6 Antibody. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody. The non-specific band shown at 25 kDa is unrelated to the primary antibody.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 408
Claudin-6 recognizes endogenous levels of total Claudin-6 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human Claudin-6 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Tight junctions, or zonula 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 the basolateral cell surfaces. Tight junctions are composed of claudin and occludin 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. Alteration in claudin protein expression pattern is associated with several types of cancer (2,3). Claudin-1 is expressed primarily in keratinocytes (4) and normal mammary epithelial cells, but is absent or reduced in breast carcinomas and breast cancer cell lines (5,6).
Claudin-6 is a member of the CLDN family that is expressed in epithelial cell sheets. Downregulation of Claudin-6 has been reported in breast invasive ductal carcinoma associated with lymphatic metastasis which may point to a function of Claudin-6 as a tumor suppressor. Claudin-6 is reported to play a role in inhibiting malignancy of breast cancer cells by inducing apoptosis, inhibiting proliferation, and migration. Mechanisms of action of Claudin-6 have been described through various signaling pathways such as p38-MAPK, JAKs-STATs, ASK1-JNK, and other pathways (7,8). Regulation of Claudin-6 expression may occur through epigenetic mechanisms (9).
Other reports describe aberrant expression in various malignancies. (10,11). The clinical significance of Claudin-6 dysregulation has created interest in the potential for pharmaceutical intervention (12-14).
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