312
1. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4 mM
l-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate,
and 1,500 mg/L sodium bicarbonate.
2. Fetal bovine serum (FBS).
3. 100× penicillin/streptomycin solution (10,000 U/mL penicillin, 10 mg/mL streptomycin) (P/S).
4. 100× amphotericin B solution (250 μg/mL) (AmpB).
5. Phosphate-buffered saline (PBS): 5.6 mM Na 2 HPO 4 , 1.1 mM
Na 2 HPO 4 and 154 mM NaCl.
6. 0.25% trypsin/0.53 mM EDTA solution.
7. PolyJet in vitro DNA transfection reagent (from SignaGen
Laboratories).
8. CID: 6.25 μM Rapamycin (Rap, Sigma-Aldrich).
9. CID: 2.5 mM Abscisic acid (ABA, AG Scientific).
10. CID: 2.5 mM Gibberellic acid (GA 3 , Sigma-Aldrich).
11. Dimethylsulfoxide (DMSO).
12. Lysis buffer such as Thermo Scientific mPER Mammalian
Protein Extraction Reagent.
13. 100× Protease inhibitor cocktail from Sigma-Aldrich.
14. 100× Phosphatase inhibitor cocktail from Santa Cruz, cocktails A and B.
15. Bicinchoninic acid protein quantification kit such as Thermo
Scientific Micro BCA Protein Assay Kit.
1. Ni-NTA agarose resin for His6-tag based purification such as
Macherey-Nagel.
2. Buffer A: 20 mM Tris, 250 mM NaCl, 20 mM imidazole,
pH 8.
3. Buffer B: 20 mM Tris, 250 mM NaCl, 20 mM imidazole,
pH 7.
4. Kinase Assay Buffer: 20 mM MOPS, 1 mM EDTA, 10 mM
MgCl 2 , pH 7.
5. Kinase Assay Buffer: 20 mM MOPS, 1 mM EDTA, 10 mM
MgCl 2 , pH 7.
6. Appropriate kinase peptide substrate such as Srctide, Lyntide,
Kemptide, and PKAtide. These can be bought from suppliers
such as SignalChem or synthesized.
7. Adenosine triphosphate (Cold ATP, Sigma-Aldrich).
8.
32
P-labeled ATP (Hot ATP, PerkinElmer).
9. P81 phosphocellulose paper (EMD Millipore).
10. 0.85% Phosphoric acid.
2.2.2 In Cellulo
Expression in Mammalian
Cells (HEK293T)
2.3 Split Kinase
Protein Purification
2.4 RadioactivityBased Kinase Activity
Assay
Javier Castillo-Montoya and Indraneel Ghosh
1. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4 mM
l-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate,
and 1,500 mg/L sodium bicarbonate.
2. Fetal bovine serum (FBS).
3. 100× penicillin/streptomycin solution (10,000 U/mL penicillin, 10 mg/mL streptomycin) (P/S).
4. 100× amphotericin B solution (250 μg/mL) (AmpB).
5. Phosphate-buffered saline (PBS): 5.6 mM Na 2 HPO 4 , 1.1 mM
Na 2 HPO 4 and 154 mM NaCl.
6. 0.25% trypsin/0.53 mM EDTA solution.
7. PolyJet in vitro DNA transfection reagent (from SignaGen
Laboratories).
8. CID: 6.25 μM Rapamycin (Rap, Sigma-Aldrich).
9. CID: 2.5 mM Abscisic acid (ABA, AG Scientific).
10. CID: 2.5 mM Gibberellic acid (GA 3 , Sigma-Aldrich).
11. Dimethylsulfoxide (DMSO).
12. Lysis buffer such as Thermo Scientific mPER Mammalian
Protein Extraction Reagent.
13. 100× Protease inhibitor cocktail from Sigma-Aldrich.
14. 100× Phosphatase inhibitor cocktail from Santa Cruz, cocktails A and B.
15. Bicinchoninic acid protein quantification kit such as Thermo
Scientific Micro BCA Protein Assay Kit.
1. Ni-NTA agarose resin for His6-tag based purification such as
Macherey-Nagel.
2. Buffer A: 20 mM Tris, 250 mM NaCl, 20 mM imidazole,
pH 8.
3. Buffer B: 20 mM Tris, 250 mM NaCl, 20 mM imidazole,
pH 7.
4. Kinase Assay Buffer: 20 mM MOPS, 1 mM EDTA, 10 mM
MgCl 2 , pH 7.
5. Kinase Assay Buffer: 20 mM MOPS, 1 mM EDTA, 10 mM
MgCl 2 , pH 7.
6. Appropriate kinase peptide substrate such as Srctide, Lyntide,
Kemptide, and PKAtide. These can be bought from suppliers
such as SignalChem or synthesized.
7. Adenosine triphosphate (Cold ATP, Sigma-Aldrich).
8.
32
P-labeled ATP (Hot ATP, PerkinElmer).
9. P81 phosphocellulose paper (EMD Millipore).
10. 0.85% Phosphoric acid.
2.2.2 In Cellulo
Expression in Mammalian
Cells (HEK293T)
2.3 Split Kinase
Protein Purification
2.4 RadioactivityBased Kinase Activity
Assay
Javier Castillo-Montoya and Indraneel Ghosh