SKP2 Total Assay Kit Human

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. 

A549 wild type (WT) and A549-SKP2 knockout (KO) (Abcam, ab301226) cells were cultured to confluency in T175 flask and lysed in 4 mL of Lysis Buffer. Lysates were assayed for Total SKP2 using the SureFire® Ultra™ kit.

Data obtained with a 2-plate, 2-incubation protocol. Adherent cell lines were seeded at 40K/well in a 96-well plate and incubated overnight. Suspension cell lines were seeded at 400K/well in HBSS + 0.1% BSA in a 96-well plate. Cells were lysed with Lysis Buffer and assayed for SKP2 Total using the SureFire® Ultra™ kit. Equivalent to 4,000 cells/datapoint for adherent cell lines and 40,000 cells/datapoint for suspension cell lines.