Anti-phospho-Protein Kinase R (Thr446) (PKR, ds RNA-dependent Protein Kinase)

Protein kinase R (PKR) is transcriptionally induced by interferon and activated by double-stranded RNA (dsRNA). PKR inhibits translation initiation through phosphorylation of the alpha subunit of the initiation factor eIF2 (eIF2alpha) and also controls the activation of several transcription factors such as NF-kappaB, p53 and Stats. In addition, PKR mediates apoptosis induced by many different stimuli, such as LPS, TNF-alpha, viral infection and serum starvation. Activation of PKR by dsRNA results in PKR dimerization and autophosphorylation at Thr446 and Thr451 in the activation loop. Substitution of Thr451 to alanine completely inactivates PKR, while a mutant with a Thr446 to alanine substitution was partially active. PKR phosphorylates the a subunit of eukaryotic initiation factor-2 and inhibits protein synthesis. It is an important component of the interferon response to virus infection. Recently, it has been reported that PKR also phosphorylates human p53 on serine392. Applications: Suitable for use in Western Blot and Beadlyte? Peptide Specificity Assay. Other applications not tested. Recommended Dilution: Western Blot: 0.5-2.0ug/ml detects phospho-PKR in RIPA lysates from HeLa cells that had been stimulated with IFN alpha overnight followed by treatment with calyculin A for 15 minutes. Treated HeLa or untreated HeLa cell lysates were resolved by electrophoresis, transferred to nitrocellulose and probed with P3103-74D (0.5ug/ml). Proteins were visualized using an IgG (HRP) goat anti-rabbit and a chemiluminescence detection system. , Beadlyte? Peptide Specificity Assay: 1:1000-1:5000 dilutions were incubated with a cocktail of microspheres conjugated to either the phosphorylated or non phosphorylated peptides. , Optimal dilutions to be determined by the researcher. Storage and Stability: May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 12 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.