Recombinant Human Active ITK (aa 352-620) Protein, CF

• Active Kinase - Active ITK (0.1 μg/μL) diluted with Kinase Dilution Buffer IX (1X) and assayed as outlined in sample activity plot. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
• Kinase Assay Buffer III (5X) - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl₂, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
• Kinase Dilution Buffer IX (1X) - Kinase Assay Buffer III diluted at a 1:4 ratio (5X dilution) with cold water. Add fresh DTT to the aliquot prior to use to a final concentration of 50 μM.
• ADP-Glo™ Kinase Assay Kit - ATP Solution, 10 mM, ADP Solution, 10 mM, ADP-Glo™ Reagent, and Kinase Detection Reagent.
• Substrate - Myelin basic protein (MBP) diluted in 100 mM MOPS (pH 6.5) buffer to a final concentration of 0.5 mg/mL.

1. Thaw the Active ITK, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution at the desired concentration, with Kinase Dilution Buffer IX (1X), in a pre-chilled 96-well plate.
2. Prepare a Substrate/ATP mixture as follows (25 μM example):
   a. 10 mM ATP Solution: 1 μL
   b. Kinase Assay Buffer III (5X): 79 μL
   c. Substrate at 0.5 mg/mL: 80 μL
3. Transfer the following reaction components prepared in Step 2 to a 384-well opaque plate bringing the reaction volume up to 5 μL:
   a. 3 μL of diluted Active ITK
   b. 2 μL of Substrate/ATP mix as prepared in Step 2. This initiates the reaction.
4. Set up the blank control as outlined in Step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer IX (1X).
5. Incubate at ambient temperature for 40 minutes.
6. After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5 μL of ADP-Glo™ Reagent. Spin down and shake the 384-well plate. Then incubate the reaction mixture for another 40 minutes at ambient temperature.
7. Add 10 μL of the Kinase Detection Reagent to the 384-well plate and incubate the reaction mixture for another 30 minutes at ambient temperature.
8. Read the 384-well reaction plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
9. Determine the corrected activity (RLU) by removing the blank control value (see Step 4) for each sample and calculate the kinase specific activity as outlined below.
   
   
   Calculation of Specific Activity of ADP (RLU/pmol)
   From ADP standard curve, determine RLU/pmol of ADP
   
   Kinase Specific Activity (SA) (pmol/min/μg or nmol/min/mg)
   Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in min) x (Enzyme amount in μg or mg)]

Scientific Data

SDS-PAGE View Larger

The approximate molecular weight is 53 kDa and the purity is > 90%.