The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application notesIHC-P: 1/100 - 1/250. Perform heat mediated antigen retrieval using 0.01M Sodium Citrate Buffer, pH 6.0 before commencing with IHC staining protocol.
WB: 1/1000 - 1/5000. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa).
Is unsuitable for Flow Cyt, ICC or IP.
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
FunctionActivates EIF2AK2/PKR in the absence of double stranded RNA (dsRNA), leading to phosphorylation of EIF2S1/EFI2-alpha and inhibition of translation and induction of apoptosis. Required for siRNA production by DICER1 and for subsequent siRNA-mediated post-transcriptional gene silencing. Does not seem to be required for processing of pre-miRNA to miRNA by DICER1.
Involvement in diseaseDefects in PRKRA are the cause of dystonia type 16 (DYT16) [MIM:612067]. DYT16 is an early-onset dystonia-parkinsonism disorder. Dystonia is defined by the presence of sustained involuntary muscle contraction, often leading to abnormal postures. DYT16 patients have progressive, generalized dystonia with axial muscle involvement, oro-mandibular (sardonic smile) and laryngeal dystonia and, in some cases, parkinsonian features.
Sequence similaritiesBelongs to the PRKRA family. Contains 3 DRBM (double-stranded RNA-binding) domains.
DomainSelf-association may occur via interactions between DRBM domains as follows: DRBM 1/DRBM 1, DRBM 1/DRBM 2, DRBM 2/DRBM 2 or DRBM 3/DRBM3.
Post-translational modificationsPhosphorylated at Ser-246 in unstressed cells and at Ser-287 in stressed cells. Phosphorylation at Ser-246 appears to be a prerequisite for subsequent phosphorylation at Ser-287. Phosphorylation at Ser-246 and Ser-287 are necessary for activation of EIF2AK2/PKR under conditions of stress.
Western blot - Anti-PACT (PKR activating protein) / PRKRA antibody [EPR3224] (ab75749)
Predicted band size : 34 kDa
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: PACT (PKR activating protein)/PRKRA knockout HAP1 cell lysate (20 µg) Lane 3: K562 cell lysate (20 µg) Lane 4: HepG2 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab75749 observed at 36 kDa. Red - loading control, ab18058, observed at 124 kDa.
ab75749 was shown to specifically react with PACT (PKR activating protein)/PRKRA when PACT (PKR activating protein)/PRKRA knockout samples were used. Wild-type and PACT (PKR activating protein)/PRKRA knockout samples were subjected to SDS-PAGE. ab75749 and ab18058 (loading control to Vinculin) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) ab216776 secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
Western blot - PACT (PKR activating protein) / PRKRA antibody [EPR3224] (ab75749)
All lanes : Anti-PACT (PKR activating protein) / PRKRA antibody [EPR3224] (ab75749) at 1/1000 dilution
Lane 1 : K562 cell lysate Lane 2 : Jurkat cell lysate Lane 3 : HepG2 cell lysate
Lysates/proteins at 10 µg per lane.
Secondary HRP labelled goat anti-rabbit at 1/2000 dilution
Predicted band size : 34 kDa Observed band size : 34 kDa