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).

(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:

Buffer:

Your calculations:

Note: protocols

(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

Your calculations:




  mg1,000 μg nmol

nmol1,000 mLnmol __nM

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.

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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