- The AMPK expression plasmid includes open reading frames of
full-length α,β, and γsubunits of human AMPK with a
ribosome-binding site (RBS) upstream of each coding region
followed by three stop codons at the end of each coding region.
It also includes a 22-residue tag which contains six histidine
residues and a cleavage site for thrombin
(MGSSHHHHHHSSGLVPRGSMGT) at the N-terminal
end of theαsubunit to aid purification. In addition, the tricis-
tronic expression plasmid contains NcoIand XhoIsites for
ligation to pET14b expression vector. - Synthesize codon-optimized genes corresponding to humanα,
β, andγsubunits for expression in Sf-9 cells. To aid purifica-
tion, insert an additional 16-residue tag that includes six histi-
dine residues and a cleavable site for thrombin
(MHHHHHHSSGLVPRGS) at the N-terminal end of theα
subunit. - Buffers can be stored at20 or 80 C until further use.
- HDX reaction is highly pH dependent (tenfold change in
exchange rate for every unit change in pH), and small variations
in buffer and sample pH can lead to ambiguous and
Fig. 5SPR ligand binding profile of PF-06409577 to AMPKα 1 β 1 γ1. Raw data (colored traces) are displayed
for PF-06409577 injections against immobilized AMPK at the concentrations indicated. Superimposed are the
kinetic fits (black lines) permitting determination ofkon,koff, andKD
48 Ravi G. Kurumbail et al.