Western blot analysis of extracts from IGROV-1, A549, and 293 cells using Claudin-1 (D3H7C) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Confocal immunofluorescent analysis of IGROV-1 (positive, left), A549 (positive, center), and 293 (negative, right) cells using Claudin-1 (D3H7C) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 10
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted December 2010
Protocol Id: 3
Claudin-1 (D3H7C) Rabbit mAb recognizes endogenous levels of total claudin-1 protein. In both claudin-1-positive and claudin-1-negative cell lines, the antibody cross-reacts with a band of unknown origin, which migrates at approximately 120 kDa by SDS-PAGE electrophoresis.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro194 of human claudin-1 protein.
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).
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