PKR (Phospho-Thr258)Colorimetric Cell-Based ELISA Kit

Assay Genie provides a cell-based ELISA kit for the accurate detection of phosphorylated PKR (protein kinase R) levels in cell lysates. The colorimetric assay is read at 450 nm and is intended for use with human adherent cells, with a dynamic range of > 5000 cells. The kit is described as a convenient, lysate-free, high-throughput and sensitive assay for detecting PKR protein phosphorylation and expression profiles in cultured cells, and can be used to measure the relative amounts of phosphorylated PKR or to screen for the effects of various treatments, inhibitors or activators on PKR phosphorylation.

Detection follows an indirect ELISA format: target-specific primary antibodies capture PKR (Phospho-Thr258), and HRP-conjugated secondary antibodies bind the Fc region of the primary antibody, catalysing a colorimetric reaction upon substrate addition. A monoclonal antibody specific for human GAPDH is included as an internal positive control for normalising target absorbance, and a Crystal Violet whole-cell staining step is provided to determine cell density so that plating differences can be adjusted for.

The target, PKR, is encoded by the gene E2AK2 (Gene ID: 5610, UniProt ID: P19525). It is a serine/threonine protein kinase of the PEK family that, upon binding double-stranded RNA, undergoes autophosphorylation and activation, and then phosphorylates the alpha subunit of eIF2 alpha to inhibit protein synthesis initiation. It also regulates transcription factors such as NF-kappaB, p53 and Stats, and mediates apoptosis in response to stimuli including LPS, TNF-alpha, viral infection and serum starvation, with dysregulation of its phosphorylation linked to cancer, viral infections and autoimmune disorders.