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 corresponding to residues surrounding Ser25 of human Fyn. Antibodies are purified by protein A and peptide affinity chromatography.
The Src family of protein tyrosine kinases, which includes Src, Lyn, Fyn, Yes, Lck, Blk, and Hck, are important in the regulation of growth and differentiation of eukaryotic cells (1). Src activity is regulated by tyrosine phosphorylation at two sites, but with opposing effects. While phosphorylation at Tyr416 in the activation loop of the kinase domain upregulates enzyme activity, phosphorylation at Tyr527 in the carboxy-terminal tail by Csk renders the enzyme less active (2).
Fyn is a 59 kDa member of the Src family of tyrosine kinases. The carboxy terminus of Fyn shares extensive amino acid sequence homology with Src, but is very different within the amino-terminal 81 amino acid residues. The Fyn protein is synthesized and N-myristoylated on cytosolic polysomes and then rapidly targeted to the plasma membrane, where it is palmitoylated (3). The corresponding sequences surrounding Tyr416 and Tyr527 of Src are conserved in Fyn and thus may be similarly regulated by phosphorylation. Dually acetylated Fyn clusters in caveolae-like membrane microdomains and can interact with a variety of other signaling molecules. Fyn's biological functions are diverse and include signaling via the T cell receptor, regulation of brain function and adhesion mediated signaling (4, 5). Alteration of the levels of Fyn in appropriate target tissues may lead to better treatments for some related diseases.
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