with InstantBlue. After only a few minutes, bands demonstrat-
ing the presence of AMPK appear (seeNote 4).- Concentrate fractions containing AMPK using 30 kDa cutoff
spin filter concentrators. Typically this will be the peak fraction
of around 2 mL and the best ten side fractions. The total
volume will now be around 10–20 mL of eluate that will be
concentrated down to around 5 mL. However depending on
the amount of AMPK protein found and its purity
(as determined from the gel), it may not be necessary
(or feasible) to concentrate the pooled fractions, as if the
protein sample is over 3 mg/mL the protein may precipitate
upon concentration. In this case, separate gel filtration runs
(below) are performed with the best fractions. - Size exclusion chromatography. If not prepared earlier (step
1 ), equilibrate a Superdex 200 XK 16/600 column with the gel
filtration buffer. - Load the fractions containing AMPK from the nickel column
that have been pooled and concentrated to 5 mL onto the
Superdex 200 column using a flow rate of 1 mL/min. - Run the column and collect 1.5 mL fractions in 2 mL tubes or a
96 deep-well plate. Full-length AMPK protein elutes at an
elution volume of approximately 70 mL (in the region of
fraction 35–50). - Monitor the eluted protein by following the absorbance at
280 nm and run an SDS-PAGE gel as above of the peak
fractions and side fractions (Fig.1). - After gel filtration, the samples need to be handled very gently
to prevent aggregation. - Pool the fractions containing AMPK. (SeeNotes 5and 6 .)
- Measure the protein concentration of each sample using a
NanoDrop spectrometer at absorbance at 280 nm.
23.In vitro phosphorylation.To prepare the phosphorylation
reaction, add AMP and ATP to the protein separately to a
final concentration of 500μM, mixing gently by pipetting
after each addition. Then add MgCl 2 to a final concentration
of 2.5 mM. Add 1 mg of CaMKKβfor every 100 mg protein.
Leave the sample overnight 16–17 h at 25C for phosphoryla-
tion to occur. There will normally be some protein precipita-
tion after this stage, with loses of around 30% not unusual.
Day 3:24.Purification of the phosphorylated AMPK protein.Clear
the sample by filtration through a 0.45μM syringe filter.
- Then reapply the protein sample to a HisTrap FF 5 mL col-
umn, which has been pre-equilibrated with nickel A buffer,
AMPK Crystallisation 9