Subheading3.2), a relationship between “volume” and DPM is
known. Dividing the “volume” with the DPM for the 10μlof
the SA gives “volume”/DPM. Dividing the “volume” of all
samples with this factor will convert them into DPM.
- The background noise of the assay has to be subtracted from
the activities of the samples. The blind samples “precipitated”
with none or irrelevant antibody will have a small activity that is
considered as noise. For this reason, subtract the mean value of
the blind samples from all other samples.
- Because only 20μl of each sample was spotted onto the P81
filter paper, the DPM has to be corrected to represent the
whole sample. The kinase reaction took place in a mixture of
10 μl of agarose beads and 30μl of kinase reaction buffer. Then
10 μl of 1% phosphoric acid was added to stop the reaction,
bringing the total volume up to 50μl. Spotting 20μl of the
total 50μl is only 40%, which makes the total activity in the
sample 2.5 times higher than measured.
- The activity of tracer (^33 P-ATP) represents the total amount of
ATP added to the sample of which the amount incorporated
into the substrate peptide is measured. The kinase reaction
buffer has 200μM ATP in 30μl¼ 6000 pmol ATP (the
contribution of ATP from the tracer is negligible
(0.33 pmol)). These 6000 pmol ATP are represented by the
total radioactive activity in a sample theoretically being
2,220,000 DPM (measured empirically in every assay as the
SA).
- All elements are now ready for the calculation of the AMPK
activity in the samples (x):
50 μl∗Sample DPM∗6000 pmol ATP
20 μl∗Total DPM∗30min∗ 0 :25 mg
¼xpmol=min=mg
50 μl representing the total reaction volume.
20 μl representing the volume spotted onto the P81 filter
paper.
Sample DPM representing the amount of ATP incorporated
into the substrate peptide.
6000 pmolATPrepresenting the total amount of ATP in the
kinase reaction.
Total DPM representing the radioactive activity of the total
amount of ATP in the kinase reaction.
30 min representing the duration of the kinase reaction.
0.25 mg representing the total amount of lysate protein from
which AMPK was isolated (seeNote 10).
Kinase Activity of Specific Heterotrimers 223
