Recombinant Human Active ZAP70 Protein, CF

• Active Kinase - Active ZAP70 (0.1 μg/μL) diluted with Kinase Dilution Buffer X and assayed as outlined in sample activity plot. Note: These are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active ZAP70 for optimal results.
• 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 X - 40 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, 2.5 mM MnCl₂, and 0.1 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 50 μM.
• ADP-Glo™ Kinase Assay Kit - 10 mM of ATP Solution, 10 mM of ADP Solution, ADP-Glo™ Reagent, and Kinase Detection Reagent.
• Substrate - Poly (4:1 Glu:Tyr) synthetic peptide substrate diluted in 25 mM Tris-HCl to a final concentration of 1 mg/mL.
• Cofactor: 2.5 M MnCl₂ - Diluted in distilled water to a working concentration of 1 M.

1. Thaw the Active ZAP70, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution with Kinase Dilution Buffer X at the desired concentration, in a pre-chilled 96-well plate.
2. Prepare a Substrate/ATP mixture as follows (25 μM ATP example):
   a. 10 mM ATP Solution: 1 μL
   b. Kinase Assay Buffer III (5X): 78 μL
   c. Substrate at 1 mg/mL: 80 μL
   d. 1 M MnCl₂: 1 μL
3. Transfer the following reaction components prepared in Steps 1 and 2 to a 384-well opaque plate, bringing the reaction volume up to 5 μL:
   a. Diluted Active ZAP70: 3 μL
   b. Substrate/ATP (mix as prepared in Step 2). This initiates the reaction: 2 μL
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 X.
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. Then 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/minutes/μg or nmol/minutes/mg)
   Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)]

Scientific Data

SDS-PAGE View Larger

The approximate molecular weight is 96 kDa and the purity is > 90%.