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Table of Materials

ProductActivity assayBinding assayPack sizeNumber of standard wells
KinaseXX100 μgVariable
5X Kinase Buffer A1,2XX4 mL of 5X2,000 x 20 μL
Phosphospecific Antibody3X
25 μg
1 mg		4,000 x 20 μL
160,000 x 20 μL
Substrate
X

1 mg (peptide)
1 mL (poly-GT OR -GAT)
20 nmol (protein)
125,000 (except Fl-p53)5
16,700 x 20 μL
10,000 x 20 μL
Antibody Dilution Buffer4X100 mL10,000 x 20 μL
500 mM EDTAX 1 mL2,500 x 20 μL
10 mM ATPX 500 μLVariable (e.g., 5,000 x 20 μL at 100 μM ATP)
Kinase Tracer6 X25 μL in 100% DMSOVariable (e.g., 830 x 15 μL at 100 nM Tracer 236)
Anti-epitope Tag Antibody3 X25 μg 1 mg5,300 x 15 μL 210,000 x 15 μL
Control InhibitorXXKinase-specific (often staurosporine,7 PHZ1271 100 μg)N/A




  1. Kinase Buffer A is supplied as a 5X concentrated stock. Prepare a 1X solution by adding 4 mL of the 5X solution to 16 mL of ultrapure water. The 1X Kinase Buffer A is stable at room temperature. 1X Kinase Buffer A consists of 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35.

  2. Some kinases require additives such as calmodulin and Ca2+, which need to be added to the Kinase Buffer A. Specific additives can be found in individual Kinase LanthaScreen® Activity validation packets.

  3. Prior to use, the antibody tube should be centrifuged at approximately 10,000 x g for 10 minutes, and the volume needed for the assay should be carefully pipetted from the supernatant.

  4. The antibody dilution buffer does not contain EDTA. EDTA is added separately to TR-FRET dilution buffer, followed by the addition of antibody.

  5. Assays containing Fl-p53 as a substrate are run at a much higher concentration of substrate, so 1 mg will run 29,500 wells.

  6. Check the tube for the concentration of tracer. For Tracer 236, this is 50 μM in DMSO; for Tracer 178, 199, 314, or 1710, this is 25 μM in DMSO.

  7. A 1 mM stock of staurosporine can be prepared by dissolving 100 μg of staurosporine in 210 μL of DMSO. The molecular weight of staurosporine is 466.53 Da.


Note:   Kinase reaction buffer is the general buffer name used to indicate that Kinase Buffer A may also contain additives.

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

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LT129       1-Jan-2010