The Knase Assay protocal.docVIP

The Kinase Assay （following JBC，2012，Jorg Kudla） 1.??? Prepare 0.75mm 12% SDSgel as usual 2.??? Prepare 10× kinase buffer 10×buffer: 667mM Tris?HCl（pH8.0） 1000mM NaCl 50mM MnSO4 5mM CaCl2 20mM DTT 0.1mM ATP 3.??? Prepare kinase reaction system in eppendorf tube as follows: Reaction System: 10×Buffer????2.4ul Kinase??? ?? ? ?1ug Add ddH2O to 24ul 4.??? Leave at Room Temperature for 30min 5.??? Stopped by adding 8ul 6× loading buffer, and denatured 5min @ 95℃, then Centrifuged 1min at 10000rpm 6.??? loaded and electrophoresis 2h at 108V The Kinase Assay 1.??? Prepare 0.75mm 12% SDSgel as usual 2.??? Prepare 10× kinase buffer 10×buffer: 500mM Tris?HCl（PH7.6） 100mM MgCl2 10mM DTT 1mM ATP 3.??? Prepare kinase reaction system in eppendorf tube (imported) as follows: Reaction System: 10?Buffer????1.5ul Kinase??? ?? ? ?1ug Add ddH2O to 14.5ul 4.??? Add 0.5ul (5uCi) [r-32P]-ATP to reaction system in Isotopic Laboratory 5.??? Leave at Room Temperature for 30min 6.??? Stopped by adding 3ul 6× loading buffer, and denatured 5min @ 95℃, then Centrifuged 1min at 10000rpm 7.??? loaded and electrophoresis 2h at 108V 8.??? Dry the gel in Gel dryer at 80℃ for 45min 9.??? Wrapped by preservative film, and exposed to X ray film in cassette 10.??? Developed the X ray film after 10h and go for another x ray film. ?Notes: 1.??? I used the GST to be negative control by substitute equal amount of GST for the NJ49 protein. 2.??? Adding ddH2O with calculated volume before the kinase may be helpful, when prepared the reaction system From Xu J, Li H-D, Chen L-Q, Wang Y, Liu L-L, He L, and Wu W-H, 2006. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell, 125: 1347~1360 Expression Vectors, Purification of GST Fusion Proteins, and In Vitro Kinase Assays The respective coding sequences were cloned into pGEX-4T-2 (Pharmacia) to generate GST-CIPK23 (CA, constitutively active), GSTcAKT1 (the cytosolic region)