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Store at -20 0C Cat # P0501 PH Domain of Akt GFP Tagged 15875 Gaither Drive Gaithersburg, MD 20877 FAX. 301-560-4919 TEL. 301-330-5966 Toll Free. 1-(866)-918-6812 10 g / 20 l Email: [email protected] Web: www.signagenlabs.com This product is for laboratory research ONLY and not for diagnostic use Introduction: Pleckstrin homology (PH) domains are ~120 amino acid–long protein modules that were first described in pleckstrin, the major protein kinase C substrate in platelets. PH domains have since been identified in several key regulatory proteins with characteristic structural features that include two orthogonal beta sheets that form a sandwich with an a helix at the COOH terminus, and variable loops that create a highly charged surface. It has been generally accepted that PH domains provide a structural basis for the interaction of certain regulatory proteins with membranes. The search for proteins that would bind and regulate proteins via PH domains has generally been unsuccessful, although in some cases, such as the b-adrenergic receptor kinase, the region that confers regulation by G protein beta gamma subunits overlaps with the PH domain. On the other hand, evidence is accumulating to suggest that PH domains of several proteins interact with membrane phosphoinositides, and that this interaction is critical to the regulation of those proteins. The PH domains of of Akt was used for real-time labeling of PI3K activation. GFP tagged Akt PH domain was found to translocate to the membrane after PI3K was activated. The membrane accumulation of GFP-PH (Akt) was prevented by pre-treatment with PI3K inhibitors such as LY294002, wortmannin, etc. Also GFP tagged Akt PH domain was utilized as a sensitive tool to label PI-(3,4)-P2 in real-time manner. Map: cDNA of Akt PH Domain (1~167) 2007 SignaGen Laboratories Procedures to Make this Clone: The PH domains of Akt (1–167) was amplified with the Advantage Klentaq polymerase mix (CLONTECH Labs, Inc., Palo Alto, CA) from human cDNAs (marathon cDNA from brain and K562 leukemia cells; CLONTECH Labs, Inc.) with the following primer pair: Akt: 5’-GTCAGCTGGTGCATCAGAGGCTGTG-3’ 5’-CACCAGGATCACCTTGCCGAAAGTGCC-3’ The amplified DNA was subcloned into the PGEM-Easy T/A cloning vector (Promega Corp., Madison, WI) and sequenced with dideoxy sequencing. A second amplification reaction was performed from these plasmids with nested primers that contained restriction sites for appropriate cloning into the pEGFP-N1. Storage: The clone should be stored at -20 0C. This clone shipped at ambient temperature