Purify and analyze phosphoprotein interactors of the Lck SH2 domain.

The Thermo Scientific GST-Lck SH2 Domain Phosphotyrosine Capture Kit is a fast and efficient tool for capturing specific tyrosine-phosphorylated proteins from mammalian cell lysates. Lck, a member of the Src family of protein tyrosine kinases, is a critical regulator of T-lymphocyte activation and differentiation. Similar to Src, Lck's tyrosine kinase activity is regulated in a negative manner through an intramolecular interaction between Y505 and Lck's internal SH2 domain. Mutations in Lck have been identified in T-cell acute lymphoblastic leukemia. This kit enables researchers to monitor tyrosine phosphorylation events using a naturally occurring protein interaction domain. After performing the pull-down reaction, common downstream applications include Western blot and mass spectrometry.

The Src homology 2 (SH2) domain is a modular protein interaction domain composed of approximately 100 amino acids functioning to selectively bind proteins phosphorylated on tyrosine residues. SH2 domains have been identified in numerous human proteins with specific biological functions, such as adaptor proteins, kinases, phosphatases, cytoskeletal regulation, transcription, ubiquitination and phospholipid second messenger signaling. Deregulation of SH2 domain-containing proteins has been implicated in human disease including multiple cancers, diabetes and human immunodeficiency.

Lck SH2 Domain Phosphotyrosine Capture Kit Procedure

Upon addition of the supplied GST-tagged SH2 domain to a cell lysate sample, primary and secondary binding events occur according to the presence and phosphorylation or other activation states of the target receptors and interactors in the sample. The protein interaction complex is then captured and purified with beaded agarose resin using the GST-glutathione affinity system. Recovered, purified proteins can then be analyzed by Western blot, mass spectrometry or other methods to identify or quantify relative amounts of the target proteins.

Consult the "Menu of SH2 Domain Phosphotyrosine Capture Kits" for more about the Lck SH2 domain pull-down protocol, related SH2 domain kits, and other resources for studying phosphoproteins.