- This protocol is applicable
only to kinases whose activity is not altered by cell lysis or immunoprecipitation
procedures and do not require soluble cofactors for activity.
- Verify that the antibody
used quantitatively immunoprecipitates the kinase from lysates without
affecting its activity, either positively or negatively.
- Plan out the necessary
components (antibody, buffers, isotope, substrate, and P81 paper/gel
lanes) in advance by determining the number of reactions required. Plan
on including negative controls (lysate of unstimulated cells; immunoprecipitation
with non-immune antibody; sample with no enzyme) as well as a positive
control (purified active enzyme).
- Establish conditions
for the kinase assay to ensure that activity is measured well within
the linear portion of the reaction (the limiting component should be
the availability of active kinase, with substrate and ATP in excess).
The most highly active samples are the first to deplete reactants, slowing
activity in those samples.
1. Prepare cell lysate
using stimulated cells and perform immunoprecipitation according to procedures
and using buffers described in this protocol section.
2. Centrifuge down
the immunoprecipitate and remove as much of the supernatant as possible
without
disturbing the pellet.
Note: Do not boil the immunoprecipitate
after the final washing step. Keep samples on ice.
3. Prepare the solutions
for the kinase assay:
- 5 x Assay Buffer:
100 mM MOPS; 125 mM beta-glycerol phosphate, pH 7.2; 5 mM EGTA; 5 mM
sodium orthovanadate; 5 mM dithiothreitol
- Magnesium/ATP
Cocktail: 100 microM non-radioactive
ATP and 75 mM magnesium chloride in Assay Buffer (If kinase is known
to prefer manganese ions for activation, manganese chloride can be substituted
for magnesium chloride)
- [gamma-32P]
ATP: dilute stock solution
to final concentration of 1 microCi/microliter using Magnesium/ATP Cocktail.
- Substrate:
(e.g., myelin basic protein) prepare a 2 mg/ml solution using Assay
Buffer
4. Prepare reaction
buffer mixture by adding 10 microliters of each component times the number
of
assay samples plus one to a single large
tube. This will ensure uniformity of the reactants as well as
allowing
for sufficient amounts for all samples. Mix well.
5. Measure the specific
activity of the radioactive label counting a 1:10 and 1:100 dilution of
the
[gamma-32P] ATP solution in a scintillation
counter. Specific activity is expressed as cpm/pmol
(the concentration
of the radioactive ATP is trivial, so it is assumed
zero)
6. Dispense the appropriate
amount of reaction buffer mixture to each tube containing the
immunoprecipitate,
still on ice, mix well, and transfer all the tubes to a shaking water
bath set at the
correct temperature. Incubation
temperatures and times should be optimized for each kinase.
7. Termination of
the assay at the appropriate time can be carried out by adding SDS-sample
buffer if
the samples are to be electrophoresed,
or by returning the tubes to an ice bucket. When dealing with
large
numbers of samples, it is advisable to first transfer
to ice and then add any termination mix,
because of the time
between addition of termination mix to the first and last tubes.
8. Either spot samples
of 30 microliters on P81 paper or electrophorese, transfer to nitrocellulose,
and
expose to film. If using the latter procedure,
also excise radioactive bands and determine cpm.
9. Express kinase
activity quantitatively using the specific activity of the ATP solution
measured above.
When assaying a purified kinase,
express the catalytic rate in its linear range in mol phosphate
transferred
from ATP to substrate/min/mg of kinase. Highly
active kinases transfer on the order of
micromol phosphate/min/mg
of kinase.
10. When assaying
kinase activity immunoprecipitated from a lysate, it is not possible to
express activity
per mg of kinase. Instead, determine
the relative activation by subtracting counts incorporated in
assays of non-immune immunoprecipitations from gross counts
to determine net cpm. If non-immune
cpm are not insignificant,
it may be that the immunoprecipitates are contaminated with other kinases.
Using net cpm, plot results as fold-activation
over unstimulated, or as a percentage of maximal
activity.
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