314
3. Using the generated mRNA products, express proteins using a
RRL system: Mix 1 pmol of mRNA with 0.25 μL of 1 mM
amino acid mixture, 17.5 μL of RLL, 1 μL of 25-fold solution
of the desired CID (either 6.25 μM Rap, 2.5 mM ABA or
2.5 mM GA 3 in 1% DMSO), and nuclease-free water to a final
volume of 25 μL. The final DMSO concentration in the mix
will be 0.04%. Incubate at 30 °C for 90 min (see Note 7).
4. Once protein expression is complete, proceed to Subheading
3.3 for protein purification and subsequent testing of kinase
activity. If desired (optional), the expression of the split kinase
proteins can be simultaneously performed using an in vitro
protein expression reaction using
35
S-methionine to label your
protein of interest and subsequently running the samples on a
SDS-PAGE gel and detection by autoradiography. The procedure for this step is out of the scope of this chapter, and can be
found elsewhere [32].
1. Maintain HEK293T cells in DMEM media supplemented with
10% FBS, 1× P/S solution, and 1× AmpB in a 5% CO 2 incubator, at 37 °C.
2. Plate HEK293T cells at a density of 1 × 10
6
cells per well in
6-well-plates, and incubate at 37 °C for 18–24 h prior to
transfection.
3. Co-transfect cells with 1 μg of total plasmid DNA. Typically,
for split constructs, 0.5 μg of each plasmid is co-transfected
and compared to transfections with 0.5 μg of wild-type kinase
and 0.5 μg of empty vector using a PolyJet transfection reagent
or equivalent according to manufacturer protocols (see Note
8). In brief, replenish media in cells plated in 6-well-plates, 1 h
before transfection; mix DNA plasmids in 50 μL of DMEM
media, and separately mix 3 μL of PolyJet reagent in another
50 μL of DMEM media; rapidly add the PolyJet solution to
the plasmid DNA solution, mix 3–4 times, and incubate at
room temperature for 15 min. Add the resulting 100 μL of
transfection mix to each well in the 6-well plates; gently mix
the media in the 6-well plates and incubate at 37 °C for 10 h.
4. 10 h post-transfection, remove transfection complexes, and
replenish wells with DMEM/0.1% FBS/1× P/S/AmpB media
(see Note 9) containing either 80 nM rapamycin/0.1% DMSO
or 100 μM ABA/0.1% DMSO or 100 μM GA 3 /0.1% DMSO
and 0.1% DMSO as a negative control. Incubate cells with
CID for 24–36 h at 37 °C (see Note 10).
5. 34–46 h post-transfection, wash each well with 500 μL of PBS,
and lyse cells with 100 μL of lysis buffer (mPER supplemented
with 1× protease and phosphatase inhibitors, and with the
appropriate CID in activated samples, typically, 250 nM Rap,
100 μM ABA or 100 μM GA 3 . Incubate cells with mPER solu3.2.2 In Cellulo
Expression in Mammalian
Cells (HEK293T)
Javier Castillo-Montoya and Indraneel Ghosh
3. Using the generated mRNA products, express proteins using a
RRL system: Mix 1 pmol of mRNA with 0.25 μL of 1 mM
amino acid mixture, 17.5 μL of RLL, 1 μL of 25-fold solution
of the desired CID (either 6.25 μM Rap, 2.5 mM ABA or
2.5 mM GA 3 in 1% DMSO), and nuclease-free water to a final
volume of 25 μL. The final DMSO concentration in the mix
will be 0.04%. Incubate at 30 °C for 90 min (see Note 7).
4. Once protein expression is complete, proceed to Subheading
3.3 for protein purification and subsequent testing of kinase
activity. If desired (optional), the expression of the split kinase
proteins can be simultaneously performed using an in vitro
protein expression reaction using
35
S-methionine to label your
protein of interest and subsequently running the samples on a
SDS-PAGE gel and detection by autoradiography. The procedure for this step is out of the scope of this chapter, and can be
found elsewhere [32].
1. Maintain HEK293T cells in DMEM media supplemented with
10% FBS, 1× P/S solution, and 1× AmpB in a 5% CO 2 incubator, at 37 °C.
2. Plate HEK293T cells at a density of 1 × 10
6
cells per well in
6-well-plates, and incubate at 37 °C for 18–24 h prior to
transfection.
3. Co-transfect cells with 1 μg of total plasmid DNA. Typically,
for split constructs, 0.5 μg of each plasmid is co-transfected
and compared to transfections with 0.5 μg of wild-type kinase
and 0.5 μg of empty vector using a PolyJet transfection reagent
or equivalent according to manufacturer protocols (see Note
8). In brief, replenish media in cells plated in 6-well-plates, 1 h
before transfection; mix DNA plasmids in 50 μL of DMEM
media, and separately mix 3 μL of PolyJet reagent in another
50 μL of DMEM media; rapidly add the PolyJet solution to
the plasmid DNA solution, mix 3–4 times, and incubate at
room temperature for 15 min. Add the resulting 100 μL of
transfection mix to each well in the 6-well plates; gently mix
the media in the 6-well plates and incubate at 37 °C for 10 h.
4. 10 h post-transfection, remove transfection complexes, and
replenish wells with DMEM/0.1% FBS/1× P/S/AmpB media
(see Note 9) containing either 80 nM rapamycin/0.1% DMSO
or 100 μM ABA/0.1% DMSO or 100 μM GA 3 /0.1% DMSO
and 0.1% DMSO as a negative control. Incubate cells with
CID for 24–36 h at 37 °C (see Note 10).
5. 34–46 h post-transfection, wash each well with 500 μL of PBS,
and lyse cells with 100 μL of lysis buffer (mPER supplemented
with 1× protease and phosphatase inhibitors, and with the
appropriate CID in activated samples, typically, 250 nM Rap,
100 μM ABA or 100 μM GA 3 . Incubate cells with mPER solu3.2.2 In Cellulo
Expression in Mammalian
Cells (HEK293T)
Javier Castillo-Montoya and Indraneel Ghosh