Materials and Reagents

1. Recombinantly produced substrate of interest
   For this study, full-length murine Oct4 was cloned into a mammalian expression vector containing a T3 promoter sequence and a carboxy-terminal 6X His – TEV – 3X FLAG epitope tage.
   
2. TnT Coupled Reticulocyte Lysate Systems (Promega Corporation, catalog number: L5010 )
   
3. Expression vector (with Sp6, T3, or T7 promoter sequence) containing protein of interest (Test Substrate) wild-type, putative mutant, empty vector control. You will also need a vector containing a previously confirmed (and published) target substrate if one is not commercially available to use as a control for the kinase assay.
   
4. Transcend tRNA (Promega Corporation, catalog number: L5061 )
   
5. RNaseOUT (Life Technologies, catalog number: 10777019 )
   
6. Cell line which exhibits activity for kinase of interest
   
7. SDS polyacrylamide gel
   
8. Polyvinyl difluoride membrane (PVDF) (Bio-Rad Laboratories, catalog number: 162-0177 )
   
9. Primary Antibody to Test and Control Substrates
   1. Akt (total) (Cell Signaling Technology, catalog number: 4685 )
      
   2. Phospho-Akt Substrate Antibody (Cell Signaling Technology, catalog number: 10001 )
      
   3. Gsk3 (total) (Cell Signaling Technology, catalog number: 5676 )
      
10. Kinase agonist (if required) to augment kinase activity
    This protocol uses Ro-31-8220 (Sigma-Aldrich, catalog number: R136-5MG ) as an Akt agonist
    
11. Non-radioactive Akt Kinase Assay Kit (Cell Signaling Technology, catalog number: 9840 )
    1. Immobilized Phospho-Akt (Ser473) (D9E) Rabbit mAb (Bead Conjugate)
       
    2. Phospho-GSK-3 (Ser21/9) (37F11) Rabbit mAb
       
    3. GSK-3 Fusion Protein at 0.5 mg/ml
       
    4. 10 mM ATP (50 µl)
       
12. Bead conjugated primary antibody or primary antibody and Protein A/G Agarose (Pierce Antibodies)
    
13. Phenyl methlsulfonyl fluoride (PMSF) (Sigma-Aldrich)
    
14. 1x Cell Lysis Buffer (see Recipes)
    
15. 1x Kinase Buffer (see Recipes)
    
16. 3x SDS Sample Buffer (see Recipes)
    

Equipment

1. Mini-Western/Transfer Apparatus (Bio-Rad Laboratories)
   
2. 10 cm plates
   
3. Cell scraper
   
4. 1.5 ml microfuge tube
   
5. Refrigerated microfuge
   
6. Microfuge tube rotator
   
7. Heat blocks set to 30 and 95 °C
   

Procedure