LanthaScreen® Eu Kinase Binding Assays

• (3.1)   3X compound: Prepare the 100X serial dilutions of compound as described in the “Things to know before starting” section. From these master dilutions of inhibitor in 100% DMSO, prepare 3X intermediate dilutions. An example using two columns of a 96-well plate is shown. The 96-well plate is used only as a convenient vessel for preparing the intermediate dilutions (Figure 12).
  
  
  
  

  Intermediate dilution guide
  Step 1	Add 162 μL 1X Buffer A to 2 columns of a 96-well plate
  Step 2	Transfer 5 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.
  Step 3	Mix, either with a plate shaker or 20 μL multichannel pipette.



• (3.2) Transfer 3X compounds to assay plate: An example using three replicates is shown. An 8-channel pipette is used to transfer 5 μL of the intermediate 3X dilution in the 96-well plate to the 384-well plates as shown in Figure 12. 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 384-well plate. A 16-point dilution series is created.


• (3.3) Positive controls: Add 5 μL of the highest 3X compound concentration to each well in the top half of column 4 (rows A–H, see Figure 13).

• (3.4) Negative controls: Prepare a 3% solution of DMSO by adding 6 μL of DMSO to 194 μL of 1X Kinase Buffer A. Add 5 μL to each well in the bottom half of column 4 (rows I–P, see Figure 13). The final concentration of DMSO in all of the wells should be 1%.


• (3.5) 3X Tracer: Prepare 1 mL tracer solution in 1X Kinase Buffer A at 3X the desired final assay concentration. In the Src example, the detailed protocol states that the K_d for Tracer 236 was determined to be 178 nM, and a tracer concentration of 100 nM was chosen for the experiment. Under these conditions, a high Z’-factor of 0.72 was obtained while keeping the tracer concentration close to the K_d value. Tracer 236 is supplied at 50 μM.

To prepare 3X Tracer 236:

Stock: 50 μM = 50,000 nM
1X tracer = 100 nM (from detailed protocol)
3X tracer = 300 nM

Sample calculation for 1,000 μL:

			1X tracer =100nM			3X tracer = 300 nM
	V1	x	C1	=	V2	x	C2
			[Initial]				[Final 3X]
Tracer 236	V1	x	50,000 nM	=	1000 μL	x	300nM
	V1 = 6 μL



TOP
Buffer:

Your calculations:





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  	V1	x	C1	=	V2	x	C2
  			[Initial]				[Final 3X]
  Tracer 236	V1	x	____nM	=	1000 μL	x	____nM
  	V1 = _____ μL

  
  
  Note: protocols
  
  
  
  
  
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    • (3.6) 3X Kinase/3X Antibody: Prepare 3X kinase/3X antibody solution at 15 nM kinase and 6 nM antibody. In the Src example, the kinase is supplied at 0.58 mg/mL and the Eu-anti-His antibody is supplied at 0.25 mg/mL, or 1,700 nM. The kinase concentration needs to be converted from mg/mL to nM. The molecular weight for each kinase can be found on its COA.
      
      Src has a molecular weight of 62.3 kDa; therefore, 62.3 μg = 1 nmol.
      
      0.58 mg   x   1,000 μg    x     1 nmol      x   1,000 mL    =    9,300 nmol       =     9,300 nM
  
  

  	V1	x	C1	=	V2	x	C2
  			[Initial]				[Final 3X]
  Kinase	V1	x	9,300 nM	=	1,500 μL	x	15 nM
  	V1 = 2.4 μL
  Antibody	V1	x	1,700 nM	=	1,500 μL	x	6 nM
  	V1 = 5.3 μL

  
  
  
  
  
  Your calculations:
  
  
  
  
    mg1,000 μg nmol
  
  
  nmol1,000 mLnmol __nM
  
  
  
  
  
  
  
  
  
  
  
  
  
  

  	V1	x	C1	=	V2	x	C2
  			[Initial]				[Final 3X]
  Kinase	V1	x	___ nM	=	1,500 μL	x	15 nM
  	V1 = __  μL
  Antibody	V1	x	___ nM	=	1,500 μL	x	6 nM
  	V1 = __ μL

  
  
  TOP
  
  Buffer:
  
  
  • (3.7) Assay plate setup: Add 5 μL of 3X kinase/3X antibody solution and 5 μL of 3X tracer solution to each well of the assay plate (plate already contains compounds or DMSO control as shown in steps 3.2–3.4) and mix briefly, either by pipette or on a plate shaker.
  
  
  • (3.8) Cover and incubate the plate at room temperature for 60 minutes and read.
    
    Start time_____
  
  
  • (3.9) Analyze the data: Divide the acceptor/tracer emission (665 nM) by the antibody/donor emission (615 nM) to calculate the TR-FRET emission ratio. Plot the TR-FRET ratio against the log of the test compound concentration and fit to a sigmoidal dose-response curve with a variable slope. Calculate the EC₅₀ concentration from the curve. This is equal to the IC₅₀ value for the inhibitor. Representative data for a set of well-characterized kinase inhibitors are presented in Figure 14.
  
  
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    To prepare 1,500 μL:
    
  
  
  
  
  
  
  
  
  • (3.10) 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
  
  
  • (3.11) 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.
  
  

Understanding the Z’-factor

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