calibration date for the latter. If the assay is run after the
calibration date, then more of the^33 P-ATP stock is needed
and accordingly less water. If getting a too low activity in the
assay (especially in the basal samples), the amount of tracer can
be adjusted and easily increased from 1 to 2μCi per sample.
- The addition of kinase reaction buffer will stir up the agarose
beads. There is no need to shake the PCR-plate during the
incubation. Due to the low volume and the V shape of the
PCR-plate well bottoms, the agarose beads will sediment and
are very hard to stir up during incubation even at 1400 rpm
(max) in a thermo-shaker. The reaction will proceed fine with-
out stirring.
- The reason for diluting the SA is that counting it undiluted will
exceed the capacity of the Liquid Scintillation Analyzer.
- Instep 9of the Subheading3.2,20μl of the samples are
spotted onto the P81 filter paper using an eight-channel
pipette. This volume of liquid spotted onto this type of filter
paper will spread out in a circular area with a diameter of
~15 mm. If using every tip on the eight-channel pipette, the
spots would overlap, so to avoid this, only every other tip is
applied to the pipette spotting four samples at a time. Firstly,
the samples from the wells in column 1 row A, C, E, and G are
spotted; followed by column 1 row B, D, F, and H; and so
on. The columns will be the same on the filter paper as in the
PCR-plate, but the rows will be arranged differently, which of
cause has to be sorted out when quantifying the spots on the
phosphor imager (seestep 17of the Subheading3.2).
- The agarose beads are included in the 20μl spotting volume.
Put the pipette tip into the well(s) (four at a time), but not all
the way to the bottom (pressing the pipette tip through the
sedimented agarose could clot the tip), and gently mix up and
down three to four times stirring up the agarose. Transfer and
spot onto the P81 filter paper. As the total volume in the wells
is 50μl, there is enough for a double determination; however
our experience is that the assay variation is not lying in the
spotting step.
- Collect the first wash in a container and store it to let the
isotope decay before pouring it into the drain.^33 P has a half-
life of 25 days and^32 P 14 days. This is not necessary for the last
two washes as more than 90% of the isotope is in the first wash.
- There is no need for quench curve correction, because 10μlof
watery^33 P solution in 2 ml Ultima Gold gives practically no
quenching, so CPM equals DPM. If the assay is run in single
Eppendorf tubes and the researcher doesn’t have access to a
phosphor imager, then it is possible to spot on single pieces of
P81 filter paper (~1.51.5 cm). These can after washing be
Kinase Activity of Specific Heterotrimers 227
