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Determine the IC50 of a control compound


Materials required


Product or reagent Cat. No. (size) for Src assay
1 μg/mL Kinase
Prepared in Step 1.2  (Detailed assay procedure)
5X Kinase Buffer APrepared in Step 1.1  (Detailed assay procedure)
Kinase Tracer 236PV5592 (25 μL in 100% DMSO)
Eu anti-His AntibodyPV5596 (25 μg)
Master staurosporine dilution seriesPrepared prior to Step 1.1 (Compound serial dilutions)

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: The detailed protocol for Src replaces Eu-anti-His with Eu-streptavidin and biotin-anti-His. For simplicity, this protocol uses Eu-anti-His only.

Note:   Antibody centrifugation is required to remove aggregates whose Tb or Eu donor signals can disrupt data analysis.

Quick reference protocol

Binding Assay—15 μL final
Step 1
Add 5 μL intermediate dilution of 3X test compound.
Step 2Add 5 μL of 3X kinase/3X antibody mixture.
Step 3Add 5 μL of 3X tracer.
Step 4Mix, cover, and read plate after 1 hour.

Final Assay conditions


Binding Assay
(15 μL final)             
Test compound
Dilution series

Kinase5 nM (typical, check protocol)

Anti-tag Antibody2 nM

Tracer~Kd*

Buffer A
1X

*~Kd for Src is 100 nM.
TOP

Detailed Assay Procedure

  • (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 Kd 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 Kd 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            

    Buffer:

    Your calculations:




  •  

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

      V1 = _____ μL            


      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

        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            


      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 * (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

    • (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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LT129       1-Jan-2010