LanthaScreen® Kinase Activity Assays

1. Determine the EC₈₀ of kinase at the ATP K_m 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 K_m.

• (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
  Kinase	V1 = 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 K_m) = 200 nM
  2X (ATP K_m) = 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
  ATP	V1	x	10,000 μM	=	1,500 μL	x	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 K_m) =  ____  μM (from validation packet)
  2X (ATP K_m) =  ____  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
  ATP	V1	x	10,000 μM	=	1,500 μL	x	____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]
  EDTA	V1	x	500 mM	=	1,750 μL	x	20 mM
  	V1 = 70 μL
  pY20 Antibody	V1	x	3,300 nM	=	1,750 μL	x	4 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]
  EDTA	V1	x	500 mM	=	1,750 μL	x	20 mM
  	V1 = 70 μL
  pY20 Antibody	V1	x	____ nM	=	1,750 μL	x	4 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  =    (      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.       

Reagents required

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

• (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.   
  

• (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
  Kinase	V1 = _____ 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 * (σ_p + σ_n)
                                           μ_p – μ_n
  
  where σ_p = standard deviation of the positive control wells
  
  σ_n = standard deviation of the negative control wells
  
  μ_p = mean of the positive control wells
  
  μ_n = 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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