SH2 Domains

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Marligen’s SH2 domains can be used in a variety of applications such as phosphotyrosine profiling, protein-protein and protein-peptide interaction studies, and immunoprecipitation of phosphorylated proteins. These probes are valuable in differentiating phosphotyrosine profiles between normal and diseased or treated samples (Dierck et al. 2006 Nature Methods 3(9) p.737.) The SH2 domains can be used in many fields of research including signal transduction, proteomics, biomarker discovery, molecular diagnostics, and drug discovery and development.

Technology

Src-homology 2 (SH2) domains are protein molecules of ~100 amino acids that are found in a large number of proteins involved in signal transduction that bind to specific phospho (pY)-containing peptide motifs. The function of SH2 domains is to specifically recognize the phosphorylated state of tyrosine residues. SH2 domains have a conserved pocket that recognizes the phosphotyrosine residue and a second variable pocket that binds 3–6 amino acids C-terminal to the phosphotyrosine that confers specificity. SH2 domains bind to phosphopeptides with dissociation constants of ~50–500 nM. Recombinant SH2 domains are used primarily to study protein-protein interactions and for purification of tyrosine phosphorylated proteins and has applications in molecular diagnostics and biomarker discovery. Marligen offers a variety of cloned SH2 domains for signal transduction research using a variety of commonly used techniques including far-Western blotting, ELISAs, immunoprecipitation, protein-protein interaction studies and phosphopeptide screening. Examples of far-Western blots of two different cell lines probed with individual SH2-domain probes demonstrate that very distinctive phosphorylation patterns can be discerned using these reagents (Figs. 1 and 2). They are supplied as solutions at 1 ug/uL in 1XPBS/50% glycerol.

Performance Characteristics

Figures 1 and 2 demonstrate the ability of each domain probe to identify unique profiles of proteins containing phosphorylated tyrosine residues in complex mixtures of proteins derived from untreated and treated cell lines and analyzed by far-Western blotting.

Figure 1. Phosphotyrosine profiles of N54 cell lysates. N54 cells are 3T3 cells that are Abl transformed and express high levels of Abl kinase. Ten micrograms of cell lysate was run on a 4–15% polyacrylamide gel, transferred to nitrocellulose and then probed with an HRP-glutathione- SH2 conjugate by the method of Nollau and Mayer ( PNAS (2001) 98(24):13531-6.). Each SH2 domain probe gives a unique phosphotyrosine profile.

Figure 2. Phosphotyrosine profiles of cell lysates from 3T3 cells that have been treated with the potent tyrosine phosphatase inhibitor pervanadate (upper row) and untreated cells (lower row). Pervanadate is an irreversible inhibitor of several tyrosine phosphatases and treatment with pervanadate leads to rapid accumulation of tyrosine phosphorylated proteins. In this figure one can see dramatic differences in the levels of tyrosine phosphorylation between treated and untreated cells. In several cases, the profiles obtained with the pervanadate-treated cells are very similar to the profiles obtained with the Abl-transformed N54 cells shown in Figure 1. Ten micrograms of cell lysate was run on a 4-15% polyacrylamide gel, transferred to nitrocellulose and then probed with an HRP-glutathione-SH2 domain conjugate by the method of Nollau and Mayer ( PNAS (2001) 98(24):13531-6.)