- The time and temperature for an IP can vary a lot depending on
antibody affinity, considerations for protein complex stability,
and workflow. Low temperature (4C) lengthens the IP pro-
cess but maintains a higher stability for protease and phospha-
tase inhibitors as well as for protein interactions. Doing
consecutive IPs and measuring enzyme activity on each of the
IPs would challenge the number of hours in a working day if
the time for each IP was set to only a couple of hours. Our
experience is that the AMPK heterotrimeric complexes are very
stable and can endure at least three consecutive overnight IP
incubations at 4C. We have assayed theα1-,α2-, andγ3-
associated activities after 4 h and one, two, and three overnight
incubations with similar results. - The total volume of each of the firstγ3 IPs is 200μl of which
10 μl is packed agarose beads and 190μl is supernatant. By only
transferring 170μl to the next IP, protein is lost, but there
must be a safety margin to avoid transferring agarose with
bound AMPK to the next IP. The second IP (α2) will thus be
on (250μg/190μl) 170μl¼ 224 μg total protein. The total
volume of theα2 IP will be 200μl (20μl of Protein G agarose,
10 μl of antibody, and 170μl of supernatant from theγ3 IP)
like the first IP of which 190μl is supernatant. Transferring
170 μl of supernatant from the secondα2 IP to the thirdα1IP
will give this IP (224μg/190μl) 170μl¼ 200 μg total
protein. These amounts of total protein precipitated on (input)
will be used in the calculations of the specific AMPK activities
that are given as pmol ATP incorporated into the AMARA
peptide per minute of time for the kinase reaction per mg lysate
precipitated on (pmol/min/mg). - When transferring the supernatant from one IP to the next,
place the first PCR-plate on the table and use plenty of light.
Place a mirror on one side of the PCR-plate, so you can see
both the first pipette tip of the eight-channel pipette on the
side facing you and the eighth tip facing the mirror. This way
you will be sure that none of the eight pipette tips reach the
agarose at the bottom of the wells. After the transfer of all
supernatants, put the PCR-plate back on ice. - The stock solutions for preparing the kinase reaction buffer are
stored at 20 C unless otherwise indicated. We use the fol-
lowing stock concentrations: 5 mM DTT, 2 mM AMP,
1.5 mM AMARA peptide, 150 mM MgCl 2 (stored at 4C),
10 mM ATP, and^33 P-ATP (10μCi/μl) (stored at 4C). In
order to make up kinase reaction buffer for one sample (30μl),
mix 8.3μl water, 5μl DTT, 3μl AMP, 2μl AMARA peptide,
1 μl MgCl 2 , 0.6μl ATP, 0.1μl^33 P-ATP (on calibration date),
and 10μl3Assay buffer (120 mM HEPES and 240 mM
NaCl). The amount of water and^33 P-ATP depends on the
226 Jesper B. Birk and Jørgen F. P. Wojtaszewski