- When the kinase reaction has run for 30 min, remove the
PCR-plate from the heating block and stop the reaction by
adding 10μl of 1% phosphoric acid to each well at the same
pace as adding the kinase reaction buffer instep 4. - Place the P81 filter paper on a piece of table paper with wax
underside, and start spotting 20μl of the samples using an
eight-channel pipette with only every other tip applied (see
Notes 15and 16 ). - When all samples are spotted, put the P81 filter paper into a
container with 1% phosphoric acid for washing away unbound
(^33) P-ATP. Wash three times for 15 min (seeNote 17).
- After last wash in phosphoric acid, wash for 2–5 min in acetone
to displace water from the filter paper. - Let the filter paper air dry in a fume hood.
- While the P81 filter paper is drying, add 10μl of the SA to each
of three counting vials (size 6 ml) plus 2 ml Ultima Gold
scintillation cocktail, cap, and vortex. - Count the three vials (triplicate) in a Liquid Scintillation Ana-
lyzer with a program designed for^33 P (window between 2 and
249 keV, counting time 2 min or stop at 2.0 (2% sigma), and
no need for quench curve correction (seeNote 18)). These
counts (multiplied by 200) are the specific activity (SA) for the
kinase reaction buffer and what was added to all samples (see
Note 19). - When the P81 filter paper is dry, spot two times 1μl and two
times 2μl of the SA on the periphery of the paper where there
are no samples spotted. - Wrap the P81 filter paper in Vita Wrap and place it in an X-ray
exposure cassette with a phosphor-imager screen. Let it expose
for 1 or 2 days. - After exposure, scan the phosphor-imager screen in a
phosphor-imager scanner. Quantify the spots using circular
“boxes,” and apply background subtraction as local median.
The quantification will result in arbitrary “volumes” or inten-
sities that have to be converted into DPM, which will be
described in the next section.
3.3 Activity
Calculations
- Only spotting 1 and 2μl of the SA on the P81 filter paper is a
precaution to avoid saturation of the scanner (step 15of the
Subheading3.2). Spotting both 1 and 2μl gives an indication
of linearity for the phosphor-imager screen. Multiply the 1μl
spots with 10 and the 2μl spots with 5 and take the mean. This
is the arbitrary “volume” or intensity for 10μl of the SA on the
phosphor-imager screen. As the activity in 10μl of the SA has
been measured by liquid scintillation (step 14 of the
222 Jesper B. Birk and Jørgen F. P. Wojtaszewski