- The 30 kDa β-subunit has an anomalous electrophoretic
mobility. This results in theβ- and theγ-subunits (37 kDa)
being difficult to resolve on a gel despite the difference in
molecular weight. If in doubt, mass spectrometry can be used
to validate the presence of both subunits accurately. - Do not concentrate the sample at this stage as if the protein
concentration exceeds 3 mg/mL, it will precipitate during
overnight phosphorylation. - Keep a sample of unphosphorylated protein for SDS-PAGE
and mass spectrometry analysis. - In our hands, usable crystals of full-length human AMPK
(α 1 β 1 γ1) could only be produced for protein that was com-
plexed with nucleotides (AMP, ADP, or ATP) bound to theγ 1
domain, a small molecule activator bound at the interface
betweenα1 andβ1, and with the kinase in an inhibited complex
with staurosporine. Crystals could not be obtained in the
absence of either staurosporine or a small molecule ADaM
activator. However, crystals of ratα1, humanβ1, and human
γ1[ 5] were successfully produced in the absence of ADaM
activators; however, the ADaM-binding site was partially occu-
pied with SO 42 instead. Holocrystals of AMPK without acti-
vator bound but with cyclodextrin (as a mimic glycogen bound
to a different region of the GBD domain instead) have been
reported [8]. However, as they demonstrated a resolution of
4A ̊, side chains and main chain can rarely be located with
confidence.
Acknowledgments
This work was supported by the Francis Crick Institute, which
receives its core funding from Cancer Research UK, the UK Medi-
cal Research Council and the Wellcome Trust. Julia Hubbard is a
recipient of a Daphne Jackson Research Fellowship, which is
funded by the Royal Society of Chemistry and the UK Medical
Research Council. We greatly acknowledge Diamond Light Source
for access to synchrotron time under proposal MX9826.References
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