Recombinant Human Active ERK1 Protein, CF

• Active Kinase - Active ERK1 (0.1 μg/μL) diluted with Kinase Dilution Buffer 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 - 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 I diluted at a 1:4 ratio (5-fold dilution) with cold distilled water. Add fresh DTT prior to use to a final concentration of 50 μM.
• ADP-Glo^TM Kinase Assay Kit - 10 mM ATP Solution, 10 mM ADP Solution, ADP-Glo^TM Reagent, Kinase Detection Reagent.
• Substrate - Myeline basic protein (MBP) diluted in 100 mM MOPS buffer (pH 6.5) to a final concentration of 0.5 mg/mL.

1. Thaw the Active ERK1, Kinase Assay Buffer III (5x), and Substrate on ice. Prepare a 15 μL enzyme dilution using Kinase Dilution Buffer IX (1x), 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): 79 μL
   c. Substrate at 0.5 mg/mL: 80 μL
3. Transfer the following reaction components prepared in Step 1 and 2 to a 384-well opaque plate bringing the reaction volume up to 5 μL:
   a. 3 μL of diluted Active ERK1
   b. 2 μL of Substrate/ATP mix as prepared in the 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 ATP-ADP conversion 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 44 kDa and the purity is > 80%.