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
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide surrounding Leu135 of human CD44. Antibodies are purified by peptide affinity chromatography.
CD44 is a type I transmembrane glycoprotein that mediates cell-cell and cell-matrix interaction through its affinity for hyaluronic acid (HA) and possibly through other parts of the extracellular matrix (ECM). CD44 is highly polymorphic, possesses a number of alternative splice variants and undergoes extensive post-translational modifications (1,2). Increased surface levels of CD44 are characteristic of T cell activation, and expression of the protein is upregulated during the inflammatory response. Research studies have shown that interactions between CD44 and HER2 are linked to an increase in ovarian carcinoma cell growth (1-3). CD44 interacts with ezrin, radixin, and moesin (ERM), linking the actin cytoskeleton to the plasma membrane and the ECM (4-6). CD44 is constitutively phosphorylated at Ser325 in resting cells. Activation of PKC results in phosphorylation of Ser291, dephosphorylation of Ser325, disassociation of ezrin from CD44, and directional motility (4).
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