Protein Protocols Protein Protocols

Analyzing Protein Phosphorylation 605
Table 1
Commercial Source
of Multifunctional Protein Kinases
Kinase Source
Protein kinase A
a,c,d,e
Protein kinase C
a,b,c,d
Calmodulin kinase II
c
Casein kinase II
a,b,d
CDC2 kinase
d
src kinase
d
a Boehringer Mannheim.
b Calbiochem-Novabiochem.
c Sigma Chemical Co.
d TCS Biologicals Ltd.
3. Start the phosphorylation reaction by the addition of ATP, containing [γ- 32 P]-ATP (as
defined in step 1, Subheading 3.). Cap the tube and vortex briefly. Follow the phosphorylation
as a function of time by removing aliquots of the reaction at specific points in time,
every 20 s for the first minute, and then at 60-s intervals for the next 4 min.
4. Terminate the reaction by mixing the sample with an equal volume of double-strength
SDS-PAGE sample buffer at room temperature. Dispense the phosphorylation sample into
an Eppendorf tube containing an equal volume of double-strength sample buffer, cap the
tube, and vortex briefly. Store these samples at room temperature, behind appropriate
screens, until all samples have been collected.
5. Incubate all samples for 30 min at 37°C. Perform SDS-PAGE in a gel of suitable
acrylamide composition, loading a minimum of 5 µg pure target protein/lane (details of
electrophoresis in Chapter 11). Allow the dye front to migrate off the bottom of the gel
(depositing most of the radioactivity into the electrode buffer), stain with Coomassie
brilliant blue, and destain the gel (see Chapter 11).
6. Mount the gel on filter paper and cover with clingfilm. The filter paper should be wet with
unused destain solution prior to contact with the SDS-PAGE gel. Lower the gel onto the
wet filter paper slowly, and flatten to remove air bubbles. Cover with clingfilm, and dry
using a vacuum-assisted gel drier for 2 h at 90°C.
7. Once dry, an X-ray film should be placed in contact with the gel for a protracted period to
image the location of phosphoproteins. This procedure must be performed in the dark,
although light emitted by dark room safety lamps is permitted. An X-ray film is first
exposed to a conditioning flash of light from a flash gun. Hold a single piece of X-ray film
and the flash gun 75 cm apart. Set the flash gun to the minimum power output, and discharge
a single flash directly onto the film (see Note 3). Place the film on top of a clean
intensifying screen within an X-ray cassette. Take the dried SDS-PAGE gel, still sandwiched
between filter paper and clingfilm, and place it gel side down onto the X-ray film.
Do not allow the gel to move once in contact with the film, and use adhesive tape to secure
the contact. With a permanent marker pen, draw distinctive markings from the filter paper
backing of the gel onto the X-ray film to facilitate orientation of film and gel once autoradiography
is complete (see Note 4). Close the X-ray cassette, label the cassette with
experimental details, including the current date and time, and store at –70°C.