- Place each square into a scintillation vial containing 3 mL of
scintillation fluid and count them using a suitable^32 P program. - Also count duplicate or triplicate samples of the labeled ATP
used in the assays. To do this, make a 1:50 dilution of the
labeled ATP and pipette 10μL onto one or more P81 paper
squares. Air-dry these paper squares without washing with
phosphoric acid. Knowing the amount of ATP in each 10μL
aliquot (if you diluted your 1 mM stock by 50-fold this will be
200 pmol), it is then easy to calculate the specific radioactivity
in cpm/nmol. If these paper squares are counted whenever a
set of assays is performed using that batch of radioactive ATP
and the specific radioactivity recalculated, there is no need to
correct for decay of^32 P radioactivity. - Subtract the values obtained in blanks without peptide sub-
strate, divide by the specific radioactivity of the ATP in
cpm/nmol to obtain the nmol phosphate incorporated, and
then divide by the incubation time in minutes to obtain the
enzyme activity in nmol/min. It is also possible to divide by the
weight (mg) of kinase added to obtain the enzyme activity in
nmol/min/mg protein. Remember to correct for the fact that
only 15μL (assuming that was indeed the volume sampled) of
the 25μL assay had been pipetted onto the P81 paper square.
3.5 Standard
Immunoprecipitate
Kinase Assay
Prior to the assay itself, all steps should be carried out on ice or at
4 C.Steps 1– 4 relate to the binding of the antibody to protein
G-Sepharose and can be carried out in advance of the assay. It is
possible to prepare sufficient antibody bound to protein
G-Sepharose for a large number of experiments (seeNote 10).
The protocol below relates to 20 immunoprecipitates (60 assays,
2 duplicates assayed with substrate peptide, and 1 blank assayed
without peptide for each immunoprecipitate) but can be scaled up
or down according to need. This assay is also applicable when
immunoprecipitating tagged proteins expressed in mammalian
cells (seeNote 11). In this case substitute anti-AMPK antibody by
the appropriate anti-epitope antibody (seeChapter10).- Wash 120μL (packed volume) of protein G-Sepharose with
3 1 mL of IP buffer. Resuspend in 1 mL of IP buffer. This
will be sufficient for 20 immunoprecipitation reactions (see
Note 12). - Add 120μg of anti-AMPK antibody (1μg of antibody perμL
of packed protein G-Sepharose beads). Adjust the volume of
the protein G-Sepharose solution with IP buffer, such that the
beads constitute 20% of the total volume, to ensure adequate
mixing. Mix on a roller mixer for 1 h at 4C. - Centrifuge the slurry at 17,000gfor 2 min at 4C, and wash
the pellet with 31 mL of IP buffer containing 0.5 M NaCl,
78 Fiona A. Fyffe et al.