315
tion for 20 min at 4 °C. Cell lysate is then cleared at 14,000 rcf
for 10 min at 4 °C.
6. Quantitate total protein concentration in each sample using
BCA reagent, according to protocol: Mix 5 μL of each sample
with 245 μL of 10% mPER in water, and 250 μL of BCA
reagent. Incubate samples at 60 °C for 30 min and then mix
200 μL of each sample with 800 μL of water in a plastic spectrometer cuvette. Read absorbance at 562 nm and compare
with a standard curve of 0–0.05 μg BSA/μL.
7. Take 150 μg of total protein from each sample and make up
the volume to 100 μL using mPER solution. Proceed to
Subheading 3.3 for protein purification and subsequent testing
of kinase activity.
1. All of the buffers used in this procedure should be supplemented with the appropriate CID (250 nM Rap, 100 μM
ABA, or 100 μM GA 3 ) in the activated samples.
2. For in vitro translated samples, dilute the 25 μL of RLL after
translation with 75 μL of Buffer A, for a final volume of
100 μL. The in cellulo translated samples are already at a volume of 100 μL.
3. For each sample, add 5 μL of Ni-NTA agarose resin (MachereyNagel) and 100 μL of Buffer A in a microcentrifuge tube (see
Note 11), and equilibrate for 30 min at 4 °C. Spin down the
resin at 1530 rcf for 1 min and remove 75 μL of supernatant
(see Note 12).
4. Add the 100 μL of cell lysate either from in vitro or in cellulo
translation and bind to the resin for 1 h at 4 °C. Gently spin down
the resin (1530 rcf/1 min) and remove 100 μL of supernatant.
5. Wash the resin for 4 min with 100 μL each time with the following buffers: 1× Buffer A, 3× Buffer B, 1× Kinase Assay
Buffer (see Note 13).
6. After the last wash, remove a total of 120 μL of supernatant,
leaving a total volume of 10 μL in the microcentrifuge tube
(see Note 14).
1. Add 10 μL of an appropriate peptide substrate solution (see
Note 15) and incubate at RT for 30 min.
2. Add 10 μL of a 300 μM ATP solution (83.3 nCi/μL, equivalent
to adding
1
/ 24 of hot
32
P–ATP PerkinElmer BLU002250UC)
and incubate at room temperature for 4 h (see Note 16).
3. Spot the reaction mixture after incubation on P81 paper, and
then wash the paper for 3 min 3× with 500 mL of 0.85%
phosphoric acid and 1× with 500 mL of acetone.
4. Immerse the P81 paper in 10 mL of scintillation cocktail in a scintillation vial and measure counts using a scintillation counter.
3.3 Split Kinase
Protein Purification
3.4 Kinase Assay
Split-Protein Kinases
tion for 20 min at 4 °C. Cell lysate is then cleared at 14,000 rcf
for 10 min at 4 °C.
6. Quantitate total protein concentration in each sample using
BCA reagent, according to protocol: Mix 5 μL of each sample
with 245 μL of 10% mPER in water, and 250 μL of BCA
reagent. Incubate samples at 60 °C for 30 min and then mix
200 μL of each sample with 800 μL of water in a plastic spectrometer cuvette. Read absorbance at 562 nm and compare
with a standard curve of 0–0.05 μg BSA/μL.
7. Take 150 μg of total protein from each sample and make up
the volume to 100 μL using mPER solution. Proceed to
Subheading 3.3 for protein purification and subsequent testing
of kinase activity.
1. All of the buffers used in this procedure should be supplemented with the appropriate CID (250 nM Rap, 100 μM
ABA, or 100 μM GA 3 ) in the activated samples.
2. For in vitro translated samples, dilute the 25 μL of RLL after
translation with 75 μL of Buffer A, for a final volume of
100 μL. The in cellulo translated samples are already at a volume of 100 μL.
3. For each sample, add 5 μL of Ni-NTA agarose resin (MachereyNagel) and 100 μL of Buffer A in a microcentrifuge tube (see
Note 11), and equilibrate for 30 min at 4 °C. Spin down the
resin at 1530 rcf for 1 min and remove 75 μL of supernatant
(see Note 12).
4. Add the 100 μL of cell lysate either from in vitro or in cellulo
translation and bind to the resin for 1 h at 4 °C. Gently spin down
the resin (1530 rcf/1 min) and remove 100 μL of supernatant.
5. Wash the resin for 4 min with 100 μL each time with the following buffers: 1× Buffer A, 3× Buffer B, 1× Kinase Assay
Buffer (see Note 13).
6. After the last wash, remove a total of 120 μL of supernatant,
leaving a total volume of 10 μL in the microcentrifuge tube
(see Note 14).
1. Add 10 μL of an appropriate peptide substrate solution (see
Note 15) and incubate at RT for 30 min.
2. Add 10 μL of a 300 μM ATP solution (83.3 nCi/μL, equivalent
to adding
1
/ 24 of hot
32
P–ATP PerkinElmer BLU002250UC)
and incubate at room temperature for 4 h (see Note 16).
3. Spot the reaction mixture after incubation on P81 paper, and
then wash the paper for 3 min 3× with 500 mL of 0.85%
phosphoric acid and 1× with 500 mL of acetone.
4. Immerse the P81 paper in 10 mL of scintillation cocktail in a scintillation vial and measure counts using a scintillation counter.
3.3 Split Kinase
Protein Purification
3.4 Kinase Assay
Split-Protein Kinases