|Product||Activity assay||Binding assay||Pack size||Number of standard wells|
|5X Kinase Buffer A1,2||X||X||4 mL of 5X||2,000 x 20 μL|
|Phosphospecific Antibody3||X|| |
|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 Buffer4||X||100 mL||10,000 x 20 μL|
|500 mM EDTA||X||1 mL||2,500 x 20 μL|
|10 mM ATP||X||500 μL||Variable (e.g., 5,000 x 20 μL at 100 μM ATP)|
|Kinase Tracer6||X||25 μL in 100% DMSO||Variable (e.g., 830 x 15 μL at 100 nM Tracer 236)|
|Anti-epitope Tag Antibody3||X||25 μg 1 mg||5,300 x 15 μL 210,000 x 15 μL|
|Control Inhibitor||X||X||Kinase-specific (often staurosporine,7 PHZ1271 100 μg)||N/A|
- 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.
- 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 at www.invitrogen.com/lanthascreenkinase.
- 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.
- The antibody dilution buffer does not contain EDTA. EDTA is added separately to TR-FRET dilution buffer, followed by the addition of antibody.
- Assays containing Fl-p53 as a substrate are run at a much higher concentration of substrate, so 1 mg will run 29,500 wells.
- 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.
- 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.