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Overview of the optimization steps when using an Invitrogen™ kinase

  1. Determine the EC80 of kinase at the ATP Km using a serial dilution of kinase.

  2. Determine the IC50 of a control test inhibitor, such as staurosporine, at the EC80 of kinase at the ATP Km.

Reagents required for Src


Product Cat. No. (size)
Src KinaseP3044 (10 μg)
5X Kinase Buffer APV3189 (4 mL of 5X)
Tb-pY20PV3528 (25 μg)
Fl-poly-GT SubstratePV3610 (1 mL)
TR-FRET Dilution BufferPV3574 (100 mL)
Kinase Quench Buffer (500 mM EDTA)P2825 (1 mL)
10 mM ATPPV3227 (500 μL)
StaurosporinePHZ1271 (100 μg)

Important:   Prior to use, antibodies used in LanthaScreen® assays should be centrifuged at approximately 10,000 x g for 10 minutes. Pipet the solution needed for the assay carefully by aspirating from the top of the solution.

Note:   Antibody centrifugation is required to remove aggregates whose Tb or Eu donor signals can disrupt data analysis.
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Step 1: Determine the EC80 of kinase at the apparent ATP Km

Quick reference protocol

LanthaScreen® Kinase Activity Assay—20 μL final
Kinase Reaction—10 μL
Step 1Prepare 2X serial dilution series of kinase (5 μL per well).
Step 2Add 5 μL of 2X substrate/2X ATP mixture.
Step 3Mix reagents, cover, and react for 1 hour.
Detection reaction ---10 μL addition, 20 μL final volume
Step 4Add 10 μL of 2X EDTA/2X antibody mixture.
Step 5Mix, cover, and read plate after 30 minutes.

Assay conditions

Kinase reaction (10μL)                                             
>5 logs kinase concentration

200 nM substrate (typical, check individual kinase)

ATP = ATP Km

1X Kinase Buffer A + any kinase-specific additives
Detection
reaction
(20 μL final)
10 mM EDTA

2 nM phosphospecific antibody

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Detailed Assay Procedure

  • (1.1) 1X Kinase Buffer A: Prepare a 1X solution of Kinase Buffer A from the 5X Kinase Buffer A stock (PV3189) by adding 4 mL of 5X stock to 16 mL ultrapure H2O to make 20 mL. If additives are required, first dilute them in the 16 mL of ultrapure H2O.

 

  • (1.2) 2X kinase: In an appropriate tube or vial, prepare 250 μL of kinase in 1X Kinase Buffer A at 2 times the highest concentration of kinase to be tested. In the Src example, 0.5 μg/mL (500 ng/mL) was the highest concentration of kinase to be tested, and the stock concentration of kinase was 580 μg/mL.
    To prepare 2X kinase: Stock = 580 μg/mL
    1X = 0.5 μg/mL
    2X = 1 μg/mL

     V1  x  
    C1=V2   x  
        C2
       [Initial]   [Final 2X]
     V1  x  580 μg/mL=250 μL  x  1 μg/mL
    KinaseV1 = 0.43 μL      


    • Note: 1X Kinase Buffer A may be stored at room temperature for future use.
    • Note: This section is taken directly from the Src validation packet, Step 3.
    • Note: If your kinase requires additives such as calcium and calmodulin, please be sure to substitute a complete kinase reaction buffer for 1X Kinase Buffer A in the activity assay protocol.
    • Note: Centrifuge antibodies once at the beginning of the day and store on ice, being careful not to disturb the pellet.
    • Note: Src is a highly active kinase and therefore has a relatively low 2X starting concentration of 1 μg/mL. To get a full dose response curve for some kinases, a 2X starting concentration of up to 10 μg/mL may be required.
    • Note: Mix kinase solutions by inversion or pipetting up and down; do not vortex.
    • Note: If V1 is less than 2 μL, perform an intermediate 10-fold dilution. We suggest taking 2.0 μL kinase and add in it to 18 μL 1X Kinase Buffer A to make 20 μL of diluted kinase.

    V1 is less than 2 μL, so perform a 10-fold dilution by adding 2 μL of kinase to 18 μL of 1X Kinase Buffer A. Use 10 x 0.43 μL, or 4.3 μL, of the diluted kinase solution.
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    Buffer: 250 – 4.3 μL = 245.7 μL 1X Kinase Buffer A

    Your calculations:

    Kinase: _____Stock = ______μg/mL
    1X = 0.5 μg/mL (from validation packet)
    2X = 1 μg/mL

    To prepare 250 μL:


     V1  x  
    C1=V2   x  
        C2
       [Initial]   [Final 2X]
     V1  x  ____μg/mL=250 μL  x  1 μg/mL
    Kinase   V1 = _____ μL   


    V1 is less than 2 μL, so perform a 10-fold dilution by adding 2 μL of kinase to 18 μL of 1X Kinase Buffer A. Use 10 x or _______  μL of the diluted kinase solution.
    Buffer: 250 μL –  ____  μL =  _____  μL 1X Kinase Buffer A

    Add the calculated volume of kinase to the appropriate volume of 1X Kinase Buffer A. For the Src example, 4.3 μL of the kinase diluted 10-fold was added to 245.7 μL 1X Kinase Buffer A to prepare a 1 μg/mL (1,000 ng/mL). This will also be used in Step 2.2

    Keep the diluted kinase on ice until needed.




    Figure 6.
    Two-fold serial kinase dilution guide.



  • (1.3)  2X kinase serial dilution:   In a low-volume 384-well plate, fill each well in columns 1–3, rows 2–16 (B through P) with 5 μL of 1X Kinase Buffer A. Place 10 μL of the kinase dilution as prepared in step (1.2) in the top well of each column, and then perform a 2-fold serial dilution of the kinase down the plate by removing 5 μL of kinase from the well in row A, adding this to the well in row B, mixing, and repeating with the next well below. Discard 5 μL of solution from the well in row P, so that each well contains 5 μL of kinase solution.
  • (1.4) 2X substrate/2X ATP:  In an appropriate container, prepare 1 mL of a solution of substrate and ATP in 1X Kinase Buffer A at 2 times the final concentration of each reagent desired in the assay.

    If 1,000 μL or more of a solution is prepared in a plastic reagent reservoir (trough), then the next addition step can be performed with a multichannel pipette.

    To prepare 2X Substrate/2X ATP:

    Fl-poly-GT stock = 30 μM = 30,000 nM
    1X (ATP Km) = 200 nM
    2X (ATP Km) = 400 nM

    ATP stock = 10 mM = 10,000 μM
    1X = 7 μM (from validation packet)
    2X = 14 μM
    To prepare 1,500 μL:

     V1  x  
    C1=V2   x  
        C2
       [Initial]   [Final 2X]
     Fl-poly-
    GT
    V1  x  30,000 nM=1500 μL  x  400 nM

    V1 = 20 μL      
    ATPV1x10,000 μM=1,500 μLx 14 μM
     V1 = 2.1 μL      

    Buffer: 1,500 μL – 20 μL – 2.1 μL = 1,477.9 μL 1X Kinase Buffer A

    Your calculations:
    Fl-poly-GT stock = _____  μM = _____  nM
    1X = 200 nM
    2X = 400 nM
    ATP stock = 10 mM = 10,000 μM
    1X (ATP Km) =  ____  μM (from validation packet)
    2X (ATP Km) =  ____  μM

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    To prepare 1,500 μL:


     V1  x  
    C1=V2   x  
        C2
       [Initial]   [Final 2X]
     Fl-poly-
    GT
    V1  x  ______nM=1500 μL  x  400 nM

    V1_____μL      
    ATPV1x10,000 μM=1,500 μLx ____μM
     V1_____μL      

    Buffer: 1,500 μL – ___  μL – ___  μL = μL 1X Kinase Buffer A

    Add the calculated volume of substrate and ATP to the appropriate volume of 1X Kinase Buffer A. For the Src example, 20 μL of Poly-GT substrate and 2.1 μL of ATP was added to 1477.9 μL 1X Kinase Buffer A
  • (1.5) Start the kinase reaction: Add 5 μL of the 2X substrate/2X ATP solution prepared in Step 1.4 to each well of the assay plate.
  • (1.6) Cover the assay plate and allow reaction to proceed for 1 hour at room temperature.
    Start time _____
  • (1.7) 2X EDTA/2X Antibody: Prior to completion of the kinase reaction, prepare 1.5 mL of a solution of EDTA and Tb-labeled antibody at 2 times the desired final concentrations of each reagent in TR-FRET dilution buffer (not 1X Kinase Buffer A). The EDTA is provided at 500 mM, and the antibody concentration can be found on the tube or COA. For the Src example, the Tb-pY20 antibody was supplied at 3,300 nM.

    Although the antibody is stable in EDTA for several hours, it is sensitive to high EDTA concentrations. To retain stability, we recommend adding the concentrated EDTA to the dilution buffer, mixing the solution well, and then adding the antibody before mixing further.

    Note:
    We recommend adding the 5 μL of 2X substrate/ 2X ATP with an electronic multichannel pipette (such as a Matrix).

    Important: Centrifuge the antibody tube at approximately 10,000 x g for 10 minutes, and carefully pipet the desired volume from the top of the solution.

    To prepare 2X EDTA/2X antibody:


    EDTA stock = 500 mM (Kinase Quench Buffer)
    1X = 10 mM
    2X = 20 mM

    Tb-pY20 Antibody stock = 3.3 μM = 3,300 nM
    1X = 2 nM
    2X = 4 nM

    Sample calculation for 1,750 μL:

     V1  x  
    C1=V2   x  
        C2
       [Initial]   [Final 2X]
    EDTAV1  x  500 mM=1,750 μL  x  20 mM

    V1 = 70 μL      
    pY20
    Antibody
    V1x3,300 nM=1,750 μLx4 nM
     V1 = 2.1 μL      

    Buffer: 1,750 μL – 70 μL – 2.1 μL = 1,677.9 μL TR-FRET dilution buffer


    Your calculations—if a different antibody concentration is supplied

    EDTA stock = 500 mM (Kinase Quench Buffer)
    1X = 10 mM
    2X = 20 mM

    Tb-pY20 Antibody stock =   ____  nM
    1X = 2 nM
    2X = 4 nM

    To prepare 1,750 μL:


     V1  x  
    C1=V2   x  
        C2
       [Initial]   [Final 2X]
    EDTAV1  x  500 mM=1,750 μL  x  20 mM

    V1 = 70 μL      
    pY20
    Antibody
    V1x____ nM=1,750 μLx4 nM
     V1 = ___ μL      

    Buffer: 1,750 μL – 70 μL – ___ μL =  ____ μL TR-FRET dilution buffer
    Add 70 μL of 500 mM EDTA and ____ μL of _____  nM antibody to ____  μL
    TR-FRET Dilution Buffer.
  • (1.8) Start the detection reaction: Add 10 μL of the 2X Tb-antibody/2X EDTA solution (Detection Mix) prepared in Step 1.7 to each well of the assay plate and mix briefly, either by pipette or on a plate shaker.
  • (1.9) Cover the assay plate and incubate for 30 minutes at room temperature before reading on an appropriate plate reader.
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  • (1.10)  Analyze the data: Divide the acceptor emission (520 nm) by the antibody/donor emission (495 nm) to calculate the TR-FRET ratio. Plot the TR-FRET ratio against the log of the kinase concentration and fit the data to a sigmoidal dose response curve with a variable slope. Calculate the EC80 concentration from the curve.



 The following equation can be used with GraphPad™ Prism® software, where F = 80 and H = the hill slope of the curve:

EC=    (      F         1/HEC50
                  
100-F   


Alternatively, the amount of kinase needed to elicit an 80% change in TR-FRET response may be estimated from a visual inspection of the curve. It is important that future experiments to determine the IC50 value of an inhibitor be performed at or slightly below the EC80 concentration of the kinase determined from this graph.






Figure 7. 
Example of kinase titration at ATP Km. The EC80 value determined from the example data is ~10 ng/mL kinase. Based on this result, 10 ng/mL kinase is used to determine inhibitor IC50 values when performing the assay at ATP Km.       

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Step 2: Determine the IC50 of a Control Inhibitor


Reagents required

Reagent Preparation outlined at
1X Kinase Buffer AStep 1.1
1 μg/mL kinaseStep 1.2
2X substrate/2X ATPStep 1.3
Detection mix (2X EDTA/2X antibody)Step 1.4
Master test compound dilution series(prior to Step 1.1)


Note:  If your kinase requires additives such as calcium and calmodulin, please be sure to substitute a complete kinase reaction buffer for 1X Kinase Buffer A in the activity assay protocol.

Quick reference protocol

 

LanthaScreen® Kinase Activity Assay—20 μL final 
Kinase Reaction—10 μL 
Step 1Add 2.5 μL of 4X test compound.
Step 2Add 2.5 μL of 4X kinase (4X EC80).
Step 3Add 5 μL of 2X substrate/2X ATP mixture.
Step 4Mix reagents, cover, and react for 1 hour.
Detection reaction ---10 μL addition, 20 μL final volume 
Step 4Add 10 μL of 2X EDTA/2X antibody mixture.
Step 5Mix, cover, and read plate after 30 minutes.

 

Assay conditions
 
Kinase reaction (10μL)                                                                            
Serial dilution of test compound

EC80 kinase concentration determined in Step 1

200 nM substrate (typical, check individual kinase)

ATP = ATP Km (see validation packet)
 1X Kinase Buffer A + any kinase-specific additives
Detection
reaction
(20 μL final)
10 mM EDTA
 2 nM phosphospecific antibody

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Detailed Assay Procedure

  • (2.1)  4X compound: Prepare the 100X serial dilutions of compound as described in the “Things to know before starting” section. For the Src example, the 100X maximum concentration of staurosporine is 1 mM. From these master dilutions of inhibitor in 100% DMSO, prepare 4X intermediate dilutions. An example using two columns of a 96-well plate is shown (Figure 8). The 96-well plate is used only as a convenient vessel for preparing the intermediate dilutions.   

Note:  
If you are preparing for both a LanthaScreen® Activity Assay and a LanthaScreen® Eu Kinase Binding Assay, we suggest that you put the intermediate dilution for the activity assay on the left of the 96-well plate in columns 1 and 2, and the binding assay on the right of the plate in columns 11 and 12. We will not describe or illustrate transfers from columns 11 and 12; treat them as you would columns 1 and 2.


Step 1 Add 96 μL 1X Kinase Buffer A to 2 columns of a 96-well plate.
Step 2Transfer 4 μL control inhibitor or test compound from 100X master dilution series stock, transfer from strip A to column 1 and strip B to column 2 (Figure 8).
Step 3Mix, either with a plate shaker or 20 μL multichannel pipette.


  • (2.2) Transfer 4X compounds to assay plate: An example using three replicates is shown in Figure 9. For the LanthaScreen® Kinase Activity Assay with the standard reaction volume of 10 μL, 2.5 μL of 4X compound from the intermediate dilution is transferred to the 384- well plate.  

    • An 8-channel pipette is used to transfer 2.5 μL of the intermediate dilution in the 96-well plate to the 384-well plate(s) as shown in Figure 9. Columns 1, 2, and 3 of the 384-well plate are the 3 replicates. Column 1 of the intermediate stock in the 96-well plate is transferred to alternate rows of the 384-well plate, rows A, C, E, etc. Column 2 of the intermediate stock is transferred to rows B, D, F, etc. of the 383-well plate. A 16-point dilution series is created.

  • (2.3)  Positive controls: Add 2.5 μL of the highest 4X compound concentration to wells in the top half of column 4 (rows A–H).

  • (2.4)  Negative controls: Add 2.5 μL 4% DMSO (from well H2 of the intermediate dilution series) to each well in the bottom half of column 4 (rows I–P). The final concentration of DMSO in all wells should be 1%.

  • (2.5)   4X EC80 kinase: Using the kinase dilution prepared in Step 1.2, as your stock, prepare a 1 mL solution of kinase in 1X Kinase Buffer A at 4X the EC80 concentration. In the Src example, 10 ng/mL kinase was determined to be the EC80, so 4X EC80 is 40 ng/mL. The concentration of the diluted kinase stock prepared in Step 1.2 is 1,000 ng/mL.

    To prepare 4X kinase:
    Stock = 1 μg/mL = 1,000 ng/mL (from Step 1.2)
    1X (EC80) = 10 ng/mL (from Step 1.10)
    4X (EC80) = 40 ng/mL
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    To prepare 1,000 μL:

     V1  x  
    C1=V2   x  
        C2
       [Initial]
      [Final 4X]
    Kinase
    V1  x  
    1000 ng/mL=1000 μL  x  
    40 ng/mL

    V1 = 40 μL      


    Buffer: 1,000 μL – 40 μL = 960 μL 1X Kinase Buffer A

    Your calculations:
    Kinase : Stock = ng/mL (from Step 1.2)
    1X (EC80) = ng/mL (from Step 1.10)
    4X (EC80) = ng/mL
    To prepare 1,000 μL:


     V1  x  
    C1=V2   x  
        C2
       [Initial]
      [Final 4X]
     V1  x  
    1000ng/mL=1000 μL  x  
    ___ng/mL
    KinaseV1 = _____ μL   
       


    Buffer: 1,000 μL – ____μL = ____μL 1X Kinase Buffer A

    Add the calculated volume of kinase to the appropriate volume of 1X Kinase Buffer A. For the Src example, 40 μL of 1,000 ng/mL kinase was added to 960 μL 1X Kinase Buffer A as calculated in the table above.

  • (2.6)  Add 4X kinase to assay plate: Add 2.5 μL of the 4X kinase solution prepared in Step 2.5
    to each well of the assay plate.

  • (2.7)  Start the kinase reaction: Add 5 μL of the 2X substrate/2X ATP solution prepared in Step
    1.4 to each well of the assay plate and mix briefly, either by pipette or on a plate shaker.

  • (2.8)  Cover the assay plate and allow reaction to proceed for 1 hour at room temperature. Start time____

  • (2.9)  Add 10 μL of the 2X Tb-antibody/2X EDTA solution prepared in Step 1.7 to each well of the assay plate.

  • (2.10) Cover the assay plate and incubate for 30 minutes at room temperature before reading on an appropriate plate reader.

  • (2.11) Analyze the data: Divide the acceptor emission (520 nm) by the antibody/donor emission (495 nm) to calculate the TR-FRET ratio. Plot the TR-FRET ratio against the log of the test compound concentration and fit the data to a sigmoidal dose response curve with a variable slope. Representative data are shown in Figure 10. Calculate the EC50 concentration from the curve. This is equal to the IC50 value for the inhibitor.

  • (2.12) Calculate the Z’-factor: Using the equation from Zhang et al., calculate the Z’-factor using your negative and positive control wells in column 4.

    Z’-factor    =   1   -     3 * (σp + σn)
                                             μp – μn


    where σp = standard deviation of the positive control wells

    σn = standard deviation of the negative control wells

    μp = mean of the positive control wells

    μn = mean of the negative control wells

  • (2.13) Assess the data quality: Assays are qualified by the IC50 of the control inhibitor and by the statistical significance of the assay window as measured by a parameter known as the Z’-factor. An assay is passing if the IC50 of the control inhibitor is within ±½ log and is generally considered robust if Z’-factor on the assay window is greater than 0.5.
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LT129       1-Jan-2010