PKR Phospho (Thr446) 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. 

Data obtained with a 2-plate, 2-incubation protocol. THP-1 cells were seeded in a 12-well plate at 250,000 cells/well in medium containing 100 nM PMA and incubated for 24 hours. After 24 hours of pretreatment, the THP-1 differentiated macrophages were treated with 250 ng/mL of IFNa, IFNB or IFNy for a further 24 hours. After treatment, cells were washed with HBSS and lysed with Lysis Buffer. PKR Phospho (Thr446 and Thr451) and eIF2a Phospho (Ser51) levels were evaluated using respective SureFire® Ultra™ kits. Equivalent to approximately 25,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. HeLa cells were seeded in a 96-well plate at 20,000 cells/well in complete medium and incubated overnight. The cells were pretreated with 10 ng/mL IFNγ for 24 hours, then treated with Calyculin A at the indicated concentrations for 30 minutes. After treatment, the cells were lysed with 100 μL of Lysis Buffer and assayed for PKR Phospho (Thr446) using the SureFire® Ultra™ kit. Equivalent to approximately 2,000 cells/datapoint.

Specificity of the Phospho (Thr446) PKR assay was assessed by a peptide competition assay. Phospho (Thr446 and Thr451) and Non-Phospho PKR peptides were serially diluted into a fixed concentration of THP1 positive control lysate. Lysates were then assayed using the SureFire® Ultra™ PKR Phospho (Thr446) kit.