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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