Human and Mouse Phospho-CDK2 (Thr160) Detection Kit

The AlphaLISA™ SureFire® Ultra™ assay enables the rapid and sensitive detection of total and phosphorylated cellular proteins. AlphaLISA™ assays utilize two bead types: Acceptor Beads and Donor Beads. The Acceptor Bead is coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody which is labeled with a CaptSure™ tag. The Donor Bead is coated with streptavidin to capture the biotinylated antibody.  

In the presence of a target protein, the two target-specific antibodies bring Donor and Acceptor Beads into close proximity. When the Donor Beads are activated by a laser (680 nm), singlet oxygen is transferred to the Acceptor Bead leading to the production of an Alpha signal. The amount of light emission (615 nm) from the Acceptor Bead is directly proportional to the amount of target protein present in the sample. If an Acceptor Bead is not in close proximity (i.e. 200 nm) of a Donor bead, little to no signal is produced over background. 

The assay can be executed in a 1-plate or 2-plate assay protocol (Refer to Manual for more details).  

1-plate assay protocol: culturing of cells, treatment, lysis and assay are performed in a single well, enabling miniaturization in high throughput screening programs. 

2-plate assay protocol: cells are cultured and treated in a 96-well culture plate and lysates are then transferred into a separate plate for assay. This format allows the evaluation of multiple targets from a single lysate. 

Data obtained with a 2-plate, 2-incubation protocol. MCF7 cells were seeded at 20K cells/well in a 96 well plate and incubated overnight. Cells were treated with 5 μM Ly294002 for 2 hours and further stimulated with Neuregulin1 at the indicated concentrations for 24 hours. Both treatments were performed in serum free media. Cells were lysed with Lysis Buffer and assayed separately for Phospho (Thr160) and Total CDK2 using respective SureFire® Ultra™ kits. Equivalent to approximately 2,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. THP1 cells were harvested and seeded at 200K cells/well in a 96 well plate. Cells were treated with Palbociclib and the indicated concentrations for 24 hours. Cells were spun down at 300 RCF for 5 minutes and washed once with HBSS + 0.1% BSA. Cells were lysed with Lysis Buffer and assayed for various targets using respective SureFire® Ultra™ kits. Equivalent to approximately 4,000 cells/datapoint.