EIF2AK2 Antibody, Biotin conjugated
Description
Molecular and Functional Characteristics
Target protein: EIF2AK2 (Uniprot ID: P19525), also known as PKR, is a serine/threonine kinase activated by double-stranded RNA during viral infections. It phosphorylates EIF2S1 to inhibit protein synthesis and regulate apoptosis .
Antibody specifics:
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Conjugate: Biotin enables streptavidin-HRP binding in assays like ELISA and Western blot (WB) .
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Immunogen: Epitopes vary by product, including regions such as AA 251-360/551 or AA 329-551 .
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Host species: Primarily rabbit (polyclonal) or mouse (monoclonal) .
Applications in Research
The biotin conjugate enhances detection sensitivity in multiple techniques:
Research Findings and Validation
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Viral response studies: EIF2AK2 antibodies confirmed upregulated kinase activity during influenza A infection in murine models .
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Apoptosis regulation: Phospho-specific antibodies (e.g., anti-T446) validated EIF2AK2’s role in stress-induced cell death .
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Tissue specificity: High expression observed in hematopoietic tissues (spleen, thymus) compared to non-hematopoietic organs .
Quality Control and Validation
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Specificity: Verified via siRNA knockdown and peptide blocking in WB .
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Cross-reactivity: No significant reactivity with non-human primate or avian orthologs reported .
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Lot consistency: Suppliers provide batch-specific data sheets ensuring reproducibility .
Limitations and Considerations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- We demonstrated the activation of the PKR pathway in CADASIL PMID: 30073405
- These results establish that PKR regulation through stress-induced TRBP phosphorylation is an important mechanism ensuring cellular recovery and preventing apoptosis due to sustained PKR activation. PMID: 29348664
- Auto-phosphorylation represses PKR activity. PMID: 28281686
- The finding that zebularine upregulates CYP gene expression through DNMT1 and PKR modulation sheds light on the mechanisms controlling hepatocyte function and thus may aid in the development of new in-vitro systems using high-functioning hepatocytes PMID: 28112215
- Multiple studies identified PKR as a crucial component of the host defense mechanism against viruses. The dynamic nature of PKR's structure allows it to interact with viral and many cellular molecules that ultimately affect the function of target molecules and downstream components of their pathways. [review] PMID: 29716441
- High PKR expression is associated with Colorectal Cancer Cell Invasiveness. PMID: 30275201
- The data demonstrate that E3 promotes F1 expression by blocking activation of the double-stranded RNA-activated protein kinase R (PKR). PMID: 29997208
- Findings indicate that MSI1 plays a leading role in stress granule formation that grants cancer stem cell properties and chemoresistant stress granules in GBM, in response to stressful conditions via the PKR/eIF2alpha signaling cascade. PMID: 29486283
- Here, the authors report that LRP16 selectively interacts and activates double-stranded RNA-dependent kinase (PKR), and also acts as scaffolds to assist the formation of a ternary complex of PKR and IKKbeta, prolonging the polymers of ADP-ribose (PAR)-dependent nuclear factor kappa B (NF-kappaB) transactivation caused by DNA-damaging agents and confers acquired chemoresistance. PMID: 28820388
- These data suggest that even a modest increase in expression of this weak PKR antagonist is sufficient to enable RhCMV replication in human cells. PMID: 29263260
- Activation of PKR by TNF-alpha mRNA element enables PKR phosphorylation. PKR phosphorylation on Ser51 is necessary and sufficient for efficient splicing of TNF-alpha mRNA. PMID: 28683312
- PKR is co-opted by EV-A71 via viral protease 3C-mediated proteolytic activation to facilitate viral replication. PMID: 28702377
- Findings suggest a novel role for PKR in lung cancer cells as a mediator of radiation resistance possibly through translocation of the protein product to the nucleus. PMID: 27203671
- a novel, positive role for PKR activation and eIF2alpha phosphorylation in human globin mRNA splicing, is reported. PMID: 28374749
- Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated ablation of double-stranded RNA (dsRNA)-activated protein kinase R (PKR) restored p53 responses while boosting hepatitis C virus replication, showing that p53 inhibition results directly from viral activation of PKR. PMID: 28442604
- Gene silencing studies showed that the suppression of immunoproteasome induction is essentially dependent on protein kinase R (PKR). Indeed, the generation of a strictly immunoproteasome-dependent cytotoxic T lymphocyte epitope was impaired in in vitro processing experiments using isolated 20S proteasomes from HCV-infected cells and was restored by the silencing of PKR expression. PMID: 27833096
- data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2alpha/SG axis and enhances virus production during virus lytic infection. PMID: 29084250
- The PKR is a key constituent of the metaflammasome and interacts directly with several inflammatory kinases, such as inhibitor kappaB (IkappaB) kinase (IKK) and c-Jun N-terminal kinase (JNK), insulin receptor substrate 1 (IRS1), and component of the translational machinery such as eIF2alpha. PMID: 26831644
- infection with New World arenaviruses JUNV and MACV, but not OW LASV, activated PKR, concomitant with elevated phosphorylation of the translation initiation factor alpha subunit of eukaryotic initiation factor 2 PMID: 28794024
- The stem-loop of noncoding RNA 886 is a structural feature not only critical for inhibiting PKR autophosphorylation, but also the phosphorylation of its cellular substrate, EIF-2alpha. PMID: 28069888
- Protein kinase R (PKR) was required for induction of stress granules (SGs) by mumps virus (MuV) infection and regulated type III IFN (IFN-lambda1) mRNA stability. PMID: 27560627
- data establish a model in which the Influenza A virus NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence PMID: 28250123
- It was established in this report that interactions between PACT, ADAR1 and HIV-1-encoded Tat protein diminish the activation of PKR in response to HIV-1 infection. PMID: 28167698
- In insulitic islets from living patients with recent-onset T1D, most of the overexpressed ISGs, including GBP1, TLR3, OAS1, EIF2AK2, HLA-E, IFI6, and STAT1, showed higher expression in the islet core compared with the peri-islet area containing the surrounding immune cells PMID: 27422384
- NF90 exerts its antiviral activity by antagonizing the inhibitory role of NS1 on PKR phosphorylation PMID: 27423063
- Crucially, Chlamydia trachomatis infection resulted in robust IRE1alpha RNAse activity that was dependent on TLR4 signaling and inhibition of IRE1alpha RNAse activity prevented PKR activation. PMID: 27021640
- the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. PMID: 26608746
- This study provides insight into the molecular pathology of Cornelia de Lange syndrome by establishing a relationship between NIPBL and HDAC8 mutations and PKR activation. PMID: 26725122
- The Newcastle disease virus-induced translation shutoff at late infection times was attributed to sustaining phosphorylation of eIF2a, which is mediated by continual activation of PKR and degradation of PP1. PMID: 26869028
- The sole essential function of cytomegalovirus TRS1 is to antagonize host PKR. PMID: 26716879
- results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells. PMID: 26698173
- Classical swine fever virus (CSFV) infection increased the phosphorylation of eukaryotic translation initiation factor (eIF)2alpha and its kinase PKR. The activation of PKR during CSFV infection is beneficial to the virus. PMID: 25899421
- these data indicate a pivotal role for PKR's protein-binding function on the proliferation of pancreatic beta cells through TRAF2/RIP1/NF-kappaB/c-Myc pathways. PMID: 25715336
- The results from this study indicate an important role of RAX/PKR association in regulating PKR activity as well as ethanol neurotoxicity PMID: 25592072
- The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. PMID: 25784705
- The data support a model in which activating RNAs induce formation of a back-to-back parallel PKR kinase dimer whereas nonactivating RNAs either fail to induce dimerization or produce an alternative, inactive dimer configuration. PMID: 26488609
- Tyrosine phosphorylated EIF2AK2 plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity. PMID: 26321373
- PKR expression correlates with inferior survival and shorter remission duration for acute myeloid leukemia patients. PMID: 26202421
- No significant association was determined between the rs2254958 EIF2AK2 polymorphism and the development of IBD, or clinical outcome. PMID: 25607115
- the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. PMID: 26231208
- Protein levels of PRKR were significantly increased in prefrontal cortex in chronic excessive alcohol use. PMID: 25704249
- Mechanism by which PK2 inhibits the model eIF2alpha kinase human RNA-dependent protein kinase (PKR) as well as native insect eIF2alpha kinases, is reported. PMID: 26216977
- G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA. PMID: 24992036
- This study demonstrates that two interferon stimulated genes, i.e. PKR and ADAR1 have opposite effects on HTLV replication in vivo. PMID: 25389016
- PKR directly interacts with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues, which inhibits Tat-mediated provirus transcription. PMID: 25653431
- Authors show that the PXXP domain within G3BP1 is essential for the recruitment of PKR to stress granules, for eIF2alpha phosphorylation driven by PKR, and for nucleating stress granules of normal composition. PMID: 25520508
- Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. PMID: 25410857
- SUMO potentiates the inhibition of protein synthesis induced by PKR in response to dsRNA. PMID: 25074923
- Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. PMID: 25297997
- Cyclophilin inhibitors reduce phosphorylation of PKR and eIF2alpha during HCV infection to allow for translation of ISG products. PMID: 24786893
Q&A
How should researchers validate the specificity of biotin-conjugated EIF2AK2 antibodies in Western blot assays?
Specificity validation requires a multi-step approach. First, compare antibody reactivity against recombinant EIF2AK2 fragments (e.g., AA 329-551) and full-length protein lysates from cell lines with known EIF2AK2 expression (e.g., HeLa, 293T) . Include knockout controls, such as CRISPR-edited EIF2AK2-null cells, to confirm the absence of off-target binding. For the biotin conjugate, pre-block membranes with streptavidin to neutralize endogenous biotin signals. A 69 kDa band should dominate in human lysates, as observed in Bosterbio’s validation . Cross-reactivity assessments across species (e.g., mouse, rat) are critical, as some antibodies exhibit restricted reactivity .
What protocols optimize EIF2AK2 detection in immunohistochemistry (IHC)?
Optimal IHC requires antigen retrieval using citrate buffer (pH 6.0) and a primary antibody dilution of 1:100–1:500, depending on tissue fixation methods. For biotin-conjugated antibodies, amplify signals using streptavidin-HRP complexes and DAB chromogens. Prioritize tissues with high EIF2AK2 expression (e.g., thymus, spleen) for positive controls. Include isotype-matched irrelevant antibodies to distinguish nonspecific binding.
How do storage conditions affect antibody performance?
Biotin-conjugated EIF2AK2 antibodies stored at -80°C retain activity for >2 years, while repeated freeze-thaw cycles >3× reduce binding capacity by 40% . Storage buffers containing 50% glycerol and 0.03% Proclin 300 prevent aggregation . For long-term studies, aliquot antibodies into single-use volumes to minimize degradation.
How can EIF2AK2 biotin conjugates be integrated into multiplex assays studying viral immune responses?
In viral infection models, pair biotinylated EIF2AK2 antibodies with fluorescent streptavidin probes (e.g., Alexa Fluor 647) and co-stain with markers of viral replication (e.g., dsRNA). Use spectral flow cytometry to resolve EIF2AK2 activation kinetics in CD14+ monocytes versus T cells. Critical controls include:
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Unconjugated competition: Pre-incubate antibodies with recombinant EIF2AK2 (90–358 AA) to block binding.
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Stress induction: Treat cells with poly(I:C) to activate PKR pathways , ensuring antibody specificity correlates with phosphorylated EIF2AK2 levels.
What experimental strategies resolve contradictions in EIF2AK2 variant functional data?
The PMC study identified conflicting variant effects: some increase kinase activity (gain-of-function), while others reduce it (loss-of-function) . To reconcile this:
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Functional complementation: Express wild-type and mutant EIF2AK2 in PKR-deficient cells and measure interferon-β production via ELISA.
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Phosphorylation profiling: Use phospho-specific antibodies (e.g., anti-pThr446) alongside biotin conjugates to quantify activation states.
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Stress assays: Expose variants to oxidative stress (H2O2) or viral mimics; gain-of-function mutants will hyperactivate stress granule formation.
How does EIF2AK2’s dual kinase activity influence experimental design in neurodegeneration models?
EIF2AK2 regulates both apoptosis (via eIF2α phosphorylation) and autophagy (mTOR suppression). In iPSC-derived neurons with DYT33-associated mutations , monitor these pathways using:
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Time-course Western blots: Track phospho-eIF2α (Ser51) and LC3-II/LC3-I ratios alongside total EIF2AK2 levels .
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Subcellular fractionation: Isolate nuclear and cytoplasmic fractions to determine if mutants mislocalize (e.g., cytoplasmic aggregation) .
Methodological Considerations Table
Why do some studies report EIF2AK2 overexpression in cancer, while others link it to tumor suppression?
This duality arises from context-dependent roles:
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Pro-survival: In glioblastoma, EIF2AK2 stabilizes HIF-1α under hypoxia .
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Pro-apoptotic: In colorectal cancer, PKR activation promotes caspase-3 cleavage .
Solution: Contextualize findings using pathway enrichment analysis (e.g., KEGG’s viral carcinogenesis pathway) and condition-specific knockdown models.
How can researchers address low antibody signal in flow cytometry?
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Enhance epitope accessibility: Use gentle fixation (4% PFA, 10 min) and permeabilization (0.1% Triton X-100).
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Signal amplification: Employ biotin-tyramide deposition (TSA kits) for low-abundance targets .
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Validate with ELISA: Compare flow cytometry mean fluorescence intensity (MFI) with ELISA OD450 readings (R² >0.9 confirms linearity) .
