- When all supernatants are transferred, seal the “α2” PCR-plate
and install it to rotate overnight at 4C as described for the
“γ3” PCR-plate insteps 10and 11. - Using the eight-channel pipette, add 180μl of IP-buffer to the
“γ3” PCR-plate and make sure to stir up the agarose beads. If
they are not stirred up by the addition of IP-buffer, seal the
plate with the silicone mat and shake it gently until they are. - Spin down the agarose for 1 min at 500gand 4C.
- Aspirate the supernatant using the eight-channel suction
device (Fig.1C) or an eight-channel pipette. Be careful not
to lose any agarose. - Repeatsteps 16– 18 once with 6Assay buffer and twice with
3 Assay buffer. - After last wash in 3Assay buffer, carefully aspirate all superna-
tant, so that only the 10μl of packed agarose beads are left in
the wells. Leave the PCR-plate on ice and continue with the
kinase reaction (below). - On Day 3, repeatsteps 12– 20 for the “α2” PCR-plate and set
up the “α1” PCR-plate for the supernatant from the “α2”
PCR-plate (step 12). - On Day 4, repeatsteps 13– 20 for the “α1” PCR-plate. The
supernatant from this plate can be discarded (step 14).
3.2 Kinase Reaction 1. While the IPs in the PCR-plate are kept on ice in the aluminum
block, prepare the kinase reaction buffer. Make up 30μl for
each sample plus a little extra (for 3–4 samples) (seeNote 12).
- Relocate to a radioisotope-classified laboratory (Class C or
higher), and work behind a Plexiglas screen. - Add the calculated amount of tracer (^33 P-ATP) to the kinase
reaction buffer. - Using a repeater pipette, add 30μl of kinase reaction buffer to
each well in a steady pace. - Place the PCR-plate on the heating block (30C) for 30 min
(seeNote 13). - Take out 15μl from the excess kinase reaction buffer and dilute
it in 985μl of water. This will be used as the specific activity
(SA) of the kinase reaction buffer (seeNote 14). - During the incubation, prepare a piece of P81 ion exchange
filter paper with pencil dots each representing one sample. The
distance between the dots (18 mm) should be twice the dis-
tance between each tip on an eight-channel pipette both verti-
cally and horizontally, meaning that the area of the filter paper
will be four times the area of the PCR-plate (seeNote 15).
Kinase Activity of Specific Heterotrimers 221