immunoprecipitated from a cell line that expresses LKB1
(HEK293, COS7). Before lysis, the user may treat the cells
with an activator of AMPK (e.g., phenformin, berberine) to
ensure maximal Thr172 phosphorylation.- Some activators may initially need to be dissolved in dimethyl
sulfoxide. - Commercially available from the MRC-PPU at Dundee:See
mrcppureagents.dundee.ac.uk/reagents-view-proteins/
609955). - If screw cap or flip-top microcentrifuge tubes containing radio-
active ATP are always spun in a microcentrifuge for a few
seconds before opening, this avoids getting radioactive con-
tamination on your gloves (which would then be spread else-
where) when the tubes are opened. - We find that expression of AMPK is greatly increased when
bacterial cells are incubated in auto-induction medium com-
pared with LB medium. Expression at 25C allows phosphor-
ylation of Ser108 on the AMPK-β subunit. We find this
essential for allosteric activation, protection against dephos-
phorylation, and promotion of phosphorylation assays
by AMP. - The in-line glutathione-containing column is to remove
CaMKK2, while the gel filtration column exchanges the buffer
and removes small molecules such as Mg.ATP. - Dialysis into buffer containing 50% glycerol reduces the vol-
ume and thus further concentrates the protein. Also, buffer
containing 50% glycerol does not freeze at 20 C, and this
enhances the stability of the protein while allowing easy
dispensing. - In our experience, antibodies bound to protein G-Sepharose
are stable for several months when stored in the fridge at 4C. - N- and C-terminal tags work well for both theαandγsub-
units; such tagged proteins form active complexes with no
obvious detriment to activity or regulation. However,
N-terminal tagging ofβsubunits prevents N-terminal myris-
toylation, while C-terminal tags appear to hinder complex
formation.
12.Important: Before pipetting protein G-Sepharose, cut approx-
imately 5 mm off pipette tips with scissors. This prevents the
Sepharose beads from getting damaged or from blocking the
pipette tip.
- AMPK used for promotion of phosphorylation assays must be
inactive. The user may use either naı ̈ve, unphosphorylated,
bacterially expressed kinase or kinase immunoprecipitated
from a cell line that does not express LKB1 (Hela, G361,
Cell-Free Assays for Regulatory Ligands 83