now contains unbroken cells and cell debris should be signifi-
cantly smaller. (SeeNote 2.)- Filter the supernatant on ice (or in the cold room) using
0.45μm syringe filters. The lysate is ready for the purification
procedure; store on ice.Once lysate is ready for purification,
proceed without delay to the nickel sepharose affinity
purification.
7.Carry out all steps in a cold room at 4 ̊C.Clean and prewash
a 5 mL nickel sepharose column with five column volumes of
elution buffer (nickel B buffer) and then five column volumes
of binding buffer (nickel A buffer). - Apply the lysate to the nickel column at a rate of 2 mL/min
using a stand-alone peristaltic pump or an A ̈KTA purification
system. - Collect the eluate as it passes through the column and label as
unbound protein. - Wash the nickel column with lysis buffer (about 200 mL) at a
rate of 2 mL/min. Collect the eluate as it passes through the
column and label as wash. - If, as in this example, you are using an A ̈KTA purification
system, connect nickel buffer A to line A and nickel buffer B
to line B. Run the buffers through the lines, B before A. - Attach the nickel sepharose column to the A ̈KTA and run
nickel A buffer through until the baseline absorbance has
been reached, that is, a flat unchanging absorbance at
280 nm is observed. Then switch pumps off but leave the
column attached. (SeeNote 3.)
Day 2: - Elute the protein from the column by running a linear gradient
of nickel A and B buffers. First prepare the fraction collector
(either 1005 mL tubes or a 96 deep-well plate). Set the
gradient to 0–100% nickel B buffer over 200 mL and start the
gradient. If using A ̈KTA set 0–100% nickel B, 100 min, at flow
rate of 2 mL/min, end volume 200 mL and collect 2 mL
fractions. Follow the absorbance at 280 nm. Normally, only
one elution peak is observed, and corresponding fractions
should be pooled. Typically select five 2 mL fractions on either
side of the peak maximum. - Visualize an aliquot of each fraction on an SDS-PAGE gel to
confirm the presence of the AMPK complex protein subunits:
mix 5μL of each fraction with 5μL SDS-PAGE sample loading
buffer, heat at 95C, and load onto a 4–12% gradient gel at
200 V for 35 min. 10μL of pre-stained protein standards are
run alongside. After running the gel, remove and stain the gel
8 Julia A. Hubbard et al.