Kinase for EC₈₀ determination:

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

To prepare 250 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 K _m) = _____μM (from validation packet)
2X = _____ μM

To prepare  ______ 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: