317
tem does not express your split kinase system efficiently, a
wheat germ extract system can also be used.
8. If this transfection conditions do not work for your particular
split kinase sensor, the so-called hard-transfection conditions
using 2 μg of total plasmid, and trypsinizing and pelleting cells
before incubation with transfection mix can be tried. Specific
procedures for hard transfection can be found for different
commercial suppliers.
9. FBS can sometimes interfere with small molecule permeability.
Thus, a 0.1% FBS media instead of standard 10% FBS is recommended at the step where the CID is added.
10. Incubation of cells with CID should be optimized as a function of time. Maximum expression levels of functional split
kinases are usually reached at 24 h after addition of CID.
11. Using smaller microcentrifuge tubes (such as 0.2 or 0.6 mL)
makes it convenient for removal of supernatant without disturbing the Ni-NTA agarose resin.
12. The 25 μL or supernatant left in the microcentrifuge tube is
necessary to perform subsequent washes without disturbing
Ni-NTA agarose resin. The excess supernatant is removed at
the end of the washes, prior to the kinase activity assay.
13. For washes, removing supernatant, adding fresh buffer, doing
a quick gentle vortex, and then incubating for 4 min before
spinning down is recommended.
14. Ni-NTA agarose resin is hygroscopic and absorb buffer and
thus at the end of the washes there is usually less than the
25 μL extra volume added at the beginning. Make sure to
remove the same amount of supernatant in all samples without
disturbing the resin, since the results for the kinase activity
assay are relative to how each sample is handled.
15. Different kinases phosphorylate different commercial peptide
substrates. Find an appropriate substrate for your split kinase
of interest and validate all protocols with the full-length or
catalytic domain only and conduct experiments at the reported
K m or independently verify K m of the parent kinase prior to
studies with the split kinase.
Acknowledgment
We thank members of the Ghosh lab, Dr. Reena Zutshi, and
Luceome Biotechnologies for helpful comments and reagents.
Indraneel Ghosh is the CSO at Luceome Biotechnologies, Tucson,
AZ. We thank the NIH (1R01GM115595-01) and NSF (CHE1506091) for supporting this research.
Split-Protein Kinases
tem does not express your split kinase system efficiently, a
wheat germ extract system can also be used.
8. If this transfection conditions do not work for your particular
split kinase sensor, the so-called hard-transfection conditions
using 2 μg of total plasmid, and trypsinizing and pelleting cells
before incubation with transfection mix can be tried. Specific
procedures for hard transfection can be found for different
commercial suppliers.
9. FBS can sometimes interfere with small molecule permeability.
Thus, a 0.1% FBS media instead of standard 10% FBS is recommended at the step where the CID is added.
10. Incubation of cells with CID should be optimized as a function of time. Maximum expression levels of functional split
kinases are usually reached at 24 h after addition of CID.
11. Using smaller microcentrifuge tubes (such as 0.2 or 0.6 mL)
makes it convenient for removal of supernatant without disturbing the Ni-NTA agarose resin.
12. The 25 μL or supernatant left in the microcentrifuge tube is
necessary to perform subsequent washes without disturbing
Ni-NTA agarose resin. The excess supernatant is removed at
the end of the washes, prior to the kinase activity assay.
13. For washes, removing supernatant, adding fresh buffer, doing
a quick gentle vortex, and then incubating for 4 min before
spinning down is recommended.
14. Ni-NTA agarose resin is hygroscopic and absorb buffer and
thus at the end of the washes there is usually less than the
25 μL extra volume added at the beginning. Make sure to
remove the same amount of supernatant in all samples without
disturbing the resin, since the results for the kinase activity
assay are relative to how each sample is handled.
15. Different kinases phosphorylate different commercial peptide
substrates. Find an appropriate substrate for your split kinase
of interest and validate all protocols with the full-length or
catalytic domain only and conduct experiments at the reported
K m or independently verify K m of the parent kinase prior to
studies with the split kinase.
Acknowledgment
We thank members of the Ghosh lab, Dr. Reena Zutshi, and
Luceome Biotechnologies for helpful comments and reagents.
Indraneel Ghosh is the CSO at Luceome Biotechnologies, Tucson,
AZ. We thank the NIH (1R01GM115595-01) and NSF (CHE1506091) for supporting this research.
Split-Protein Kinases