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Calculation worksheets for LanthaScreen® Kinase Activity Assay


Kinase for EC80 determination:

Kinase:   ____ Stock = ____ mg/mL = _____ μg/mL
1X = _____ μg/mL (from validation packet)
2X = ____   μg/mL

To prepare 250 μL:




V1          x         C1        =         V2         x             C2

                      [Initial]                                           [Final 2X]
Kinase
V1          x       μg/mL    =      250 μL     x              μg/mL

V1 = μL

If V1 is less than 2 μL, perform a 10-fold dilution by adding 2 μL of kinase to 18 μL of 1X  Kinase Buffer A and add 10 x V1.

Buffer:
250 μL – __________μL = ________μL 1X Kinase Buffer A

Add the calculated volume of kinase to the appropriate volume of 1X Kinase Buffer A. Keep the diluted kinase on ice until needed.

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.


2X Substrate/2X ATP:

You may need to convert mg/mL listed on the tube to nM. The molecular weight of the substrate is listed on the COA.

Substrate____
1X = ____ nM
2X = ____  nM

ATP Stock = 10 mM = 10,000 μM
1X (ATP Km) = _____μM (from validation packet)
2X = _____ μM

To prepare  ______ μL:



V1                x          C1              =            V2              x             C2

                           [Initial]                                                    [Final 2X]
SubstrateV1                x         _____nM         =      ____  μL       x          _____nM

V1  = _____ μL
ATPV1                x          10,000 μM    =   _____μL        x                _____μM

V1  = _____ μL


If V1 is less than 2 μL, perform a 10-fold dilution by adding 2 μL of 1X kinase to 18 μL of Kinase Buffer A and add 10 x V1.

Buffer:  ____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.

2X EDTA/2X Antibody:

EDTA Stock = 500 mM
1X = 10 mM
2X = 20 mM

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


Antibody Stock = ____ μM = ____ nM
1X = 2 nM
2X = 4 nM

To prepare ____ μL:


V1              x                     C1                =             V2            x                  C2
      
                                      [Initial]                                                          [Final2X]
EDTA V1             x                500 mM            =       ____ μL        x               20 mM

V1 =  ____μL                                                                                                    
AntibodyV1              x                ____nM            =       ____μL         x                  4 nM

V1 =  ____μL   

Buffer:    ____μL – ____μL – μL = μL TR-FRET dilution buffer

Add the calculated volume of EDTA and antibody to the calculated volume of TR-FRET
dilution buffer.


4X (EC80) Kinase for IC50 determinations:


Kinase: Stock = ng/mL (from Step 1.2)
1X (EC80) = ng/mL (from Step 1.10)
4X (EC80) = ng/mL

To prepare μL:


V1          x         C1        =         V2         x             C2

                      [Initial]                                             [Final 4X]
Kinase
V1          x     ___ng/mL    =      1,000 μL     x              μg/mL

V1 = μL

Buffer: 250 μL – ______  μL = ______  μL   1X Kinase Buffer A

Add the calculated volume of kinase to the appropriate volume of 1X Kinase Buffer A.

2X Substrate/2X ATP:

You may need to convert mg/mL listed on the tube to nM. The molecular weight of the substrate is listed on the COA.
Substrate_____
1X = ____ nM
2X = ____ nM

ATP Stock = 10 mM = 10,000 μM
1X (ATP Km) = ____ μM (from validation packet)
2X = _____  μM

To prepare ______ μL:



V1                x          C1              =            V2               x             C2

                             [Initial]                                                        [Final 2X]
SubstrateV1                x           _____nM         =   ____  μL        x                  _____nM

V1  = _____ μL
ATPV1                x          10,000 μM    =      _____μL         x                  _____μM

V1  = _____ μL


If V1 is less than 2 μL, perform a 10-fold dilution by adding 2 μL of kinase to 18 μL of 1X Kinase Buffer A and add 10 x V1.

Buffer:   _____
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

2X EDTA/2X Antibody:

EDTA Stock = 500 mM
1X = 10 mM
2X = 20 mM

Antibody Stock = μM = nM
1X = 2 nM
2X = 4 nM

To prepare μL:


V1                x          C1              =            V2               x             C2

                           [Initial]                                                        [Final 2X]
EDTAV1                x         500 nM         =     ____  μL        x                20 nM

V1  = _____ μL
AntibpdyV1                x          ____ μM    =      _____μL         x                  4 nM

V1  = _____ μL


Buffer:  
____ _ μL – _____ μL – _____ μL =  ____ μL TR-FRET dilution buffer

Add the calculated volume of EDTA and antibody to the calculated volume of TR-FRET dilution buffer.

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LT129       1-Jan-2010