- Aspirate PBS and add 0.5 ml of ice-cold reduced cell lysis buffer
to the top edge of the dish. - Using a cell scraper, collect solubilized cell lysate at the bottom
edge of the dish, and pipette into a 1.5 ml Eppendorf tube. - Vortex briefly, and clarify lysate by centrifugation at 18,000
gfor 5 min, 4C. Lysate can be flash frozen in liquid nitrogen
and stored at 80 C indefinitely or processed on glutathione
agarose as described below.
3.2 Purification
of Recombinant AMPK
- For AMPK immobilization on glutathione agarose (seeNote
12 ),dispense glutathione agarose (~20μl of a 50% slurry/
0.5 ml cell lysate) into a 1.5 ml Eppendorf tube, and pellet by
centrifugation at 3,350gfor 3 min. - Wash glutathione agarose three times by resuspension in 1 ml
of glutathione agarose wash buffer, followed by centrifugation
at 3,350gfor 3 min. - After the final centrifugation step, resuspend glutathione aga-
rose in AMPK expression cell lysate. Place on a rotating wheel
at 4C for 1 h, and pellet glutathione agarose by centrifugation
at 3,350gfor 3 min. - Wash glutathione agarose three times by resuspension in 1 ml
of glutathione agarose wash buffer, followed by centrifugation
at 3,350gfor 3 min. - For AMPK elution, after the final centrifugation step, resus-
pend in glutathione agarose elution buffer (10μl buffer/10μl
agarose), and place on rotating wheel at 4C for 5 min. Pellet
agarose by centrifugation at 3,350gfor 3 min. Remove 5μl
of the AMPK elution for Western blot analysis (Fig.1;seeNote
13 ), and dispense remaining AMPK elution into 50μl aliquots
(seeNotes 14and 15 ). Flash freeze aliquots in liquid N 2 for
long-term storage at 80 C.
GST-pulldown
IB:
pan
pan
HA
1
2
3
2
1Fig. 1Immunoblot profiles of 12 recombinant AMPK complexes expressed in COS7 cells. Bothβ1 (30.4 kDa)
andγ3 (54.3 kDa) subunits display reduced electrophoretic mobility on SDS-PAGE, relative to expected
mobility based on molecular mass
164 Jonathan S. Oakhill et al.