311
2 Materials
1. Plasmids encoding for the kinase of interest and the interacting
proteins from the desired CID system (Addgene is a good
source for plasmids). We have almost exclusively focused on
protein tyrosine kinases and the protocols reflect our bias.
2. Plasmid vector for cloning, such as pRSFDuet-1 and
pcDNA3.1(+).
3. Appropriate oligonucleotide primers to generate inserts.
4. Enzymes for cloning procedures with the necessary reaction
buffers and dNTPs. Thermostable DNA polymerase (such as
Taq, e.g., from New England BioLabs or KAPA from KAPA
Biosystems), Klenow fragment (e.g., New England BioLabs),
appropriate restriction enzymes, calf intestinal alkaline phosphatase (CIP, e.g., from New England BioLabs), DNA ligase
(such as T4 DNA ligase, e.g., from New England BioLabs).
5. Competent cells, such as E. coli XL1-Blue, and appropriate
growth media, such as Luria–Bertani agar and broth.
6. Plasmid DNA and PCR product purification kits. We typically
use Macherey-Nagel NucleoSpin Plasmid, and NucleoSpin Gel
and PCR Clean-up kits.
1. DNA polymerase such as Taq or KAPA with provided reaction
buffer and dNTPs.
2. RNA production system (such as Promega T7 RiboMAX
Large Scale RNA production system), with provided reaction
buffer and rNTPs.
3. PCR-product purification kit (e.g., from Macherey-Nagel) and
G50-microcolumns for mRNA purification (e.g., from GE
Healthcare).
4. Rabbit reticulocyte lysate (RRL) system (ours was donated
by Luceome Biotechnologies), which contains the cellular
components necessary for protein synthesis (tRNAs, amino
acids, ribosomes, initiation, elongation, and termination factors).
Commercial systems are also optimized to include an energygenerating system consisting of prequalified phosphocreatine and
phosphocreatine kinase, a mixture of tRNAs to expand the range
of mRNAs that can be translated, hemin to prevent inhibition of
initiation, and potassium acetate and magnesium acetate.
5. CID: 6.25 μM Rapamycin (Rap, Sigma-Aldrich).
6. CID: 2.5 mM Abscisic acid (ABA, AG Scientific).
7. CID: 2.5 mM Gibberellic acid (GA 3 , Sigma-Aldrich).
8. Dimethylsulfoxide (DMSO)
2.1 Plasmid
Construction
and Cloning
2.2 Split Kinase
Protein Expression
2.2.1 In Vitro Expression
in Rabbit Reticulocyte
Lysate
Split-Protein Kinases
2 Materials
1. Plasmids encoding for the kinase of interest and the interacting
proteins from the desired CID system (Addgene is a good
source for plasmids). We have almost exclusively focused on
protein tyrosine kinases and the protocols reflect our bias.
2. Plasmid vector for cloning, such as pRSFDuet-1 and
pcDNA3.1(+).
3. Appropriate oligonucleotide primers to generate inserts.
4. Enzymes for cloning procedures with the necessary reaction
buffers and dNTPs. Thermostable DNA polymerase (such as
Taq, e.g., from New England BioLabs or KAPA from KAPA
Biosystems), Klenow fragment (e.g., New England BioLabs),
appropriate restriction enzymes, calf intestinal alkaline phosphatase (CIP, e.g., from New England BioLabs), DNA ligase
(such as T4 DNA ligase, e.g., from New England BioLabs).
5. Competent cells, such as E. coli XL1-Blue, and appropriate
growth media, such as Luria–Bertani agar and broth.
6. Plasmid DNA and PCR product purification kits. We typically
use Macherey-Nagel NucleoSpin Plasmid, and NucleoSpin Gel
and PCR Clean-up kits.
1. DNA polymerase such as Taq or KAPA with provided reaction
buffer and dNTPs.
2. RNA production system (such as Promega T7 RiboMAX
Large Scale RNA production system), with provided reaction
buffer and rNTPs.
3. PCR-product purification kit (e.g., from Macherey-Nagel) and
G50-microcolumns for mRNA purification (e.g., from GE
Healthcare).
4. Rabbit reticulocyte lysate (RRL) system (ours was donated
by Luceome Biotechnologies), which contains the cellular
components necessary for protein synthesis (tRNAs, amino
acids, ribosomes, initiation, elongation, and termination factors).
Commercial systems are also optimized to include an energygenerating system consisting of prequalified phosphocreatine and
phosphocreatine kinase, a mixture of tRNAs to expand the range
of mRNAs that can be translated, hemin to prevent inhibition of
initiation, and potassium acetate and magnesium acetate.
5. CID: 6.25 μM Rapamycin (Rap, Sigma-Aldrich).
6. CID: 2.5 mM Abscisic acid (ABA, AG Scientific).
7. CID: 2.5 mM Gibberellic acid (GA 3 , Sigma-Aldrich).
8. Dimethylsulfoxide (DMSO)
2.1 Plasmid
Construction
and Cloning
2.2 Split Kinase
Protein Expression
2.2.1 In Vitro Expression
in Rabbit Reticulocyte
Lysate
Split-Protein Kinases