EIF2AK3 Antibody, HRP conjugated

What is EIF2AK3 and why is it an important research target?

EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3) encodes the protein known as PERK (protein kinase RNA-like endoplasmic reticulum kinase), a key regulator of ER stress. This 1116-amino acid residue protein is involved in critical cellular processes including the apoptotic pathway and angiogenesis. PERK is localized to the endoplasmic reticulum and features N-glycosylation and phosphorylation post-translational modifications. The protein is ubiquitously expressed across numerous tissue types, making it relevant for research in multiple organ systems. Other commonly used synonyms include PEK and WRS (Wolcott-Rallison Syndrome) .

How do EIF2AK3/PERK antibodies function in basic research protocols?

EIF2AK3/PERK antibodies function by binding to specific epitopes on the PERK protein, allowing researchers to detect, quantify, and/or isolate this kinase in experimental systems. For HRP-conjugated antibodies specifically, the horseradish peroxidase enzyme directly attached to the antibody enables direct visualization when appropriate substrates are added, eliminating the need for secondary antibody incubation steps. These antibodies can be applied in various techniques including Western blotting, immunohistochemistry (IHC), immunocytochemistry (ICC), enzyme-linked immunosorbent assays (ELISA), and immunoprecipitation (IP), depending on the specific validation of each antibody product .

What are the main differences between PERK-A and PERK-B haplotypes when studying antibody responses?

PERK-A and PERK-B represent the two most common haplotypes of PERK, distinguished by specific single-nucleotide variants (SNVs). The PERK-B haplotype (formed by minor alleles of rs867529, rs13045, and rs1805165) has been associated with increased risk for multiple disorders affecting both peripheral tissues and the central nervous system. Research indicates potential functional differences between these haplotypes, with PERK-B hypothesized to exhibit increased PERK activity both in vitro and in vivo, particularly under acute ER stress conditions. When designing experiments with PERK antibodies, researchers should consider which epitopes are being targeted and whether these regions contain polymorphisms that distinguish between these haplotypes, as this may affect antibody binding and experimental outcomes .

How should I optimize Western blot protocols for EIF2AK3/PERK detection using HRP-conjugated antibodies?

For optimal Western blot detection of EIF2AK3/PERK using HRP-conjugated antibodies, implement the following methodology:

• Sample preparation: Given PERK's large size (approximately 125 kDa), use fresh samples and include protease inhibitors to prevent degradation.

• Gel selection: Employ a low-percentage (6-8%) SDS-PAGE gel for adequate resolution of this high-molecular-weight protein.

• Transfer: Utilize wet transfer for at least 90-120 minutes at constant amperage to ensure complete transfer of large proteins.

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody incubation: For HRP-conjugated PERK antibodies, dilute according to manufacturer recommendations (typically 1:1000 to 1:5000) and incubate overnight at 4°C.

• Washing: Perform 4-5 stringent washes with TBST to minimize background.

• Detection: Use enhanced chemiluminescence (ECL) substrate with exposure times adjusted according to signal strength.

• Controls: Include positive controls (cell lines known to express PERK) and consider including samples from PERK knockout models as negative controls .

What methodology should be followed when designing ELISA experiments to quantify EIF2AK3 levels?

When designing ELISA experiments for quantifying EIF2AK3 levels, implement this methodological approach:

• Sandwich ELISA format: Use a pre-coated 96-well plate with anti-EIF2AK3 antibody as the capture antibody and biotin-conjugated anti-EIF2AK3 as the detection antibody.

• Sample preparation: For cell lysates, use 100 μL maximum per well; for other liquid samples, use 50 μL maximum per well.

• Standard curve preparation: Create a dilution series covering the expected detection range (e.g., 46.875-3000 pg/mL for rat EIF2AK3).

• Assay procedure: Follow sequential incubations with standards/samples, detection antibody, HRP-Streptavidin, and TMB substrate.

• Signal detection: Measure absorbance at 450 nm after adding stop solution.

• Data analysis: Use appropriate software (e.g., CurveExpert 1.4) to generate standard curves and calculate concentrations.

• Quality control: Include intra-assay precision testing (samples tested multiple times on same plate) and inter-assay precision testing (samples tested across different plates) to validate results .

How can I determine the optimal fixation method for immunohistochemistry with EIF2AK3 antibodies?

Determining the optimal fixation method for immunohistochemistry with EIF2AK3 antibodies requires systematic testing of different conditions:

• Fixative comparison:

  • Test 4% paraformaldehyde (PFA) fixation for 24 hours

  • Compare with 10% neutral buffered formalin for 24-48 hours

  • Evaluate methanol fixation for 10 minutes at -20°C

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • HIER using Tris-EDTA buffer (pH 9.0)

  • Enzymatic retrieval with proteinase K

• Blocking conditions:

  • Test various blocking solutions (BSA, normal serum, commercial blockers)

  • Optimize blocking time (1-2 hours)

• Control experiments:

  • Include positive control tissues with known EIF2AK3 expression

  • Use tissues from PERK knockout models as negative controls

  • Perform peptide competition assays to verify antibody specificity

• Validation:

  • Compare staining patterns with published literature

  • Confirm subcellular localization (primarily ER-associated)

This systematic approach allows identification of conditions that preserve both tissue morphology and antibody epitopes .

How can I effectively use EIF2AK3 antibodies to study the unfolded protein response (UPR) in neurodegenerative disease models?

To effectively study the unfolded protein response (UPR) in neurodegenerative disease models using EIF2AK3 antibodies, implement this comprehensive approach:

• Experimental model selection:

  • Compare models relevant to diseases associated with EIF2AK3 variants (Alzheimer's, progressive supranuclear palsy)

  • Include age-matched controls to account for age-related changes in PERK activity

  • Consider transgenic models with specific PERK haplotypes (PERK-A vs. PERK-B)

• Activation state assessment:

  • Use phospho-specific antibodies to detect activated PERK (p-PERK)

  • Simultaneously assess downstream targets (p-eIF2α, ATF4, CHOP)

  • Employ dual immunofluorescence to co-localize PERK with disease-specific markers

• Temporal analysis:

  • Design time-course experiments to track UPR activation longitudinally

  • Correlate PERK activation with disease progression markers

  • Assess acute versus chronic stress responses

• Intervention studies:

  • Test PERK inhibitors/activators to modulate the UPR

  • Evaluate genetic knockdown/overexpression of PERK

  • Design rescue experiments targeting specific UPR branches

• Data interpretation:

  • Distinguish between adaptive and terminal UPR

  • Correlate findings with behavioral/functional outcomes

  • Integrate results with other UPR sensors (IRE1α, ATF6)

This methodology allows comprehensive characterization of PERK's role in neurodegenerative disease pathogenesis .

What are the critical considerations when developing a multiplexed assay including EIF2AK3 antibodies?

When developing a multiplexed assay including EIF2AK3 antibodies, address these critical considerations:

• Antibody compatibility assessment:

  • Test for cross-reactivity between primary antibodies

  • Ensure epitope accessibility when multiple targets are bound

  • Validate specificity of each antibody individually before combining

• Detection system design:

  • For fluorescence-based systems, select fluorophores with minimal spectral overlap

  • For chromogenic detection, ensure substrate compatibility

  • When using HRP conjugates, employ tyramide signal amplification for sequential detection

• Steric hindrance mitigation:

  • Target distinct cellular compartments (e.g., PERK in ER, other markers in nucleus/cytoplasm)

  • Use antibody fragments (Fab) to reduce spatial constraints

  • Optimize antibody concentrations to balance signal intensity across targets

• Technical execution:

  • Determine optimal sequential order of antibody application

  • Implement stringent blocking between steps to prevent non-specific binding

  • Include appropriate controls for each target and combination

• Signal resolution:

  • Employ spectral unmixing algorithms for fluorescence applications

  • Use multi-exposure imaging to capture signals of varying intensities

  • Implement digital image analysis to quantify co-localization

• Validation strategy:

  • Compare multiplexed results with single-plex assays

  • Confirm expected subcellular localization patterns

  • Verify physiological relevance of detected interactions

This methodical approach ensures reliable simultaneous detection of EIF2AK3 and other relevant markers .

How can I distinguish between different isoforms or phosphorylation states of EIF2AK3 in my research?

To distinguish between different isoforms or phosphorylation states of EIF2AK3, implement this specialized methodology:

• Antibody selection strategy:

  • Use phospho-specific antibodies targeting key residues (Thr980, Thr982)

  • Select antibodies recognizing specific domains (kinase domain vs. regulatory domain)

  • Employ antibodies targeting unique regions of splice variants

• Sample preparation optimization:

  • Preserve phosphorylation states by rapid processing

  • Include phosphatase inhibitors in all buffers

  • Use fractionation techniques to enrich for ER membranes

• Resolution enhancement techniques:

  • Employ Phos-tag™ SDS-PAGE to separate phosphorylated species

  • Use 2D gel electrophoresis (isoelectric focusing followed by SDS-PAGE)

  • Apply lambda phosphatase treatment to confirm phosphorylation-dependent bands

• Confirmatory approaches:

  • Implement immunoprecipitation followed by mass spectrometry

  • Use site-directed mutagenesis to validate specific phosphorylation sites

  • Apply phospho-mimetic and phospho-dead mutations to verify functional significance

• Functional validation:

  • Correlate phosphorylation state with kinase activity assays

  • Assess downstream eIF2α phosphorylation

  • Monitor effects of stress inducers (thapsigargin, tunicamycin) on phosphorylation patterns

This comprehensive approach enables precise identification and functional characterization of specific EIF2AK3 forms .

What are common causes of false-negative results when using EIF2AK3 antibodies, and how can these be addressed?

Common causes of false-negative results when using EIF2AK3 antibodies can be systematically addressed through the following methodology:

• Epitope masking issues:

  • Problem: Protein folding or fixation may obscure antibody binding sites

  • Solution: Test multiple antigen retrieval methods (heat-induced, enzymatic)

  • Validation: Compare results with antibodies targeting different epitopes

• Protein degradation:

  • Problem: PERK (125 kDa) is susceptible to proteolytic degradation

  • Solution: Use fresh samples, process rapidly, include protease inhibitor cocktails

  • Validation: Run positive controls from freshly prepared samples

• Insufficient sensitivity:

  • Problem: Low endogenous expression levels in certain tissues/conditions

  • Solution: Implement signal amplification (TSA, enhanced chemiluminescence)

  • Validation: Use enrichment techniques (immunoprecipitation) prior to detection

• Technical issues with HRP conjugates:

  • Problem: Activity loss of HRP enzyme

  • Solution: Store antibodies according to manufacturer recommendations, avoid freeze-thaw cycles

  • Validation: Include positive control for HRP activity

• Batch-to-batch variability:

  • Problem: Inconsistent antibody performance across lots

  • Solution: Validate each new lot against previous successful experiments

  • Validation: Maintain reference samples for comparison

• Unsuitable application:

  • Problem: Antibody may not be validated for specific application

  • Solution: Review validation data and select antibodies appropriate for intended use

  • Validation: Test multiple antibodies targeting different epitopes

This systematic troubleshooting approach ensures reliable detection of EIF2AK3 .

How should I validate the specificity of an EIF2AK3 antibody for my particular experimental system?

To validate the specificity of an EIF2AK3 antibody for your particular experimental system, implement this comprehensive validation protocol:

• Genetic validation:

  • Test antibody on samples with EIF2AK3 knockdown/knockout

  • Use overexpression systems with tagged EIF2AK3 constructs

  • Compare antibody performance across species if working with non-human models

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Compare results with and without peptide competition

  • Establish optimal peptide concentration for complete signal blocking

• Molecular weight verification:

  • Confirm detection at expected molecular weight (~125 kDa)

  • Check for known splice variants or cleaved forms

  • Assess cross-reactivity with related kinases (GCN2, PKR, HRI)

• Physiological relevance testing:

  • Validate expected subcellular localization (primarily ER)

  • Confirm response to known PERK activators (thapsigargin, tunicamycin)

  • Verify concurrent activation of downstream targets (p-eIF2α)

• Cross-validation with orthogonal methods:

  • Compare protein detection with mRNA expression data

  • Utilize multiple antibodies targeting different epitopes

  • Correlate with functional readouts of PERK activity

• Technical controls:

  • Include isotype controls to assess non-specific binding

  • Test secondary antibody alone to identify background issues

  • Evaluate multiple blocking agents to optimize signal-to-noise ratio

This methodical approach ensures robust antibody validation specific to your experimental system .

What internal and external controls should be included when using EIF2AK3 antibodies in quantitative assays?

When using EIF2AK3 antibodies in quantitative assays, include these comprehensive controls:

• Calibration controls:

  • Standard curve using recombinant EIF2AK3 protein (46.875-3000 pg/mL range)

  • Quality control samples at low, medium, and high concentrations

  • Reference standards traceable to international standards when available

• Experimental controls:

  • Positive tissue/cell controls (tissues with known EIF2AK3 expression)

  • Negative controls (EIF2AK3 knockout/knockdown samples)

  • Treatment controls (ER stress inducers like thapsigargin to increase PERK expression/activation)

• Technical controls:

  • Antibody-free wells to assess non-specific substrate conversion

  • Isotype controls to evaluate background binding

  • Dilution linearity tests to confirm proportional signal reduction

• Inter-assay normalization:

  • Include common reference samples across all plates/experiments

  • Use housekeeping proteins for Western blot normalization

  • Apply consistent threshold criteria for all analyses

• Precision assessment:

  • Intra-assay replicates (n=20) at low, medium, and high concentrations

  • Inter-assay replicates across multiple experimental days

  • Statistical analysis of coefficient of variation (<10% for intra-assay, <15% for inter-assay)

• Specificity controls:

  • Peptide competition controls

  • Cross-reactivity tests with related proteins

  • Antibody lot validation and bridging studies when changing lots

This comprehensive control strategy ensures reliable quantitative assessment of EIF2AK3 across experiments .

How do I interpret conflicting results between different EIF2AK3 antibodies in the same experiment?

When faced with conflicting results between different EIF2AK3 antibodies in the same experiment, apply this systematic interpretation methodology:

• Epitope mapping analysis:

  • Determine the specific regions recognized by each antibody

  • Assess whether epitopes might be differentially affected by experimental conditions

  • Consider whether epitopes span regions with known polymorphisms (rs867529, rs13045, rs1805165)

• Validation status evaluation:

  • Review validation documentation for each antibody

  • Compare application-specific validation (some antibodies work for WB but not IHC)

  • Assess species cross-reactivity documentation if relevant

• Technical parameter assessment:

  • Compare antibody formats (monoclonal vs. polyclonal, different host species)

  • Evaluate conjugation effects (unconjugated vs. HRP-conjugated)

  • Review optimal working conditions for each antibody

• Biological context consideration:

  • Assess whether conflicting results reflect different isoforms or post-translational modifications

  • Consider potential splice variants or proteolytic processing

  • Evaluate whether results reflect physiological PERK activation states

• Orthogonal method validation:

  • Implement alternative detection methods (mass spectrometry)

  • Correlate with functional assays (kinase activity)

  • Validate with genetic approaches (siRNA knockdown, CRISPR knockout)

• Resolution strategy:

  • Design peptide competition experiments with specific immunizing peptides

  • Test antibodies on samples with manipulated EIF2AK3 expression

  • Sequence the target region in your experimental system to identify potential variations

This analytical framework provides a scientific basis for reconciling apparently conflicting results .

How should PERK activation be interpreted in the context of different cellular stress conditions?

Interpreting PERK activation across different cellular stress conditions requires this nuanced analytical framework:

• Temporal activation profile analysis:

  • Acute phase (minutes to hours): Initial phosphorylation indicates adaptive response

  • Intermediate phase (6-24 hours): Sustained activation reflects ongoing stress management

  • Chronic phase (>24 hours): Persistent activation may indicate maladaptive response or failure to resolve stress

• Stress-specific response patterns:

  • ER stress (thapsigargin, tunicamycin): Primary PERK pathway activation

  • Oxidative stress: May activate PERK through indirect ER perturbation

  • Nutrient deprivation: Often activates parallel stress pathways (GCN2)

  • Viral infection: May show distinct patterns due to viral interference mechanisms

• Integrated UPR analysis:

  • Coordinate with other UPR branches (IRE1α, ATF6)

  • Assess downstream targets (p-eIF2α, ATF4, CHOP)

  • Evaluate translational repression (polysome profiles, puromycin incorporation)

• Threshold determination:

  • Identify activation thresholds that distinguish adaptive vs. terminal UPR

  • Correlate activation levels with cell fate outcomes

  • Determine cell type-specific response patterns

• Genetic background effects:

  • Consider EIF2AK3 variant impacts (PERK-A vs. PERK-B haplotypes)

  • Assess influence of genetic modifiers on UPR outcomes

  • Evaluate PERK polymorphisms in disease-specific contexts

• Intervention response characterization:

  • Measure modulation by PERK inhibitors/activators

  • Assess feedback regulation mechanisms

  • Determine recovery kinetics following stress removal

What mathematical models can be applied to analyze EIF2AK3 activity in complex biological systems?

For analyzing EIF2AK3 activity in complex biological systems, these mathematical modeling approaches can be applied:

• Ordinary differential equation (ODE) models:

  • Kinetic modeling of PERK activation/deactivation rates

  • Inclusion of phosphorylation/dephosphorylation dynamics

  • Integration with downstream signaling cascades (eIF2α, ATF4, CHOP)

  • General form: dP/dt = k₁[inactive PERK] - k₂[active PERK]

• Stochastic modeling approaches:

  • Captures cell-to-cell variability in PERK activation

  • Accounts for low-copy-number effects in single-cell analysis

  • Implementation using Gillespie algorithm or chemical Langevin equations

• Boolean network models:

  • Represents UPR components (including PERK) as ON/OFF nodes

  • Models logical relationships between pathway components

  • Efficiently captures qualitative behavior in large networks

• Bayesian inference methods:

  • Estimates PERK activity from indirect measurements

  • Incorporates prior knowledge from literature

  • Updates model parameters as new data becomes available

• Machine learning approaches:

  • Uses supervised learning to predict PERK activation from multivariate data

  • Implements unsupervised clustering to identify activation patterns

  • Employs deep learning for image-based phenotypic analysis of PERK-dependent effects

• Multi-scale modeling:

  • Links molecular PERK dynamics to cellular phenotypes

  • Connects cellular responses to tissue-level outcomes

  • Integrates temporal scales from seconds (phosphorylation) to days (adaptation)

These mathematical approaches provide rigorous frameworks for quantitative analysis of PERK behavior in complex systems .

How can EIF2AK3 antibodies be effectively utilized in neurodegenerative disease research?

For effective utilization of EIF2AK3 antibodies in neurodegenerative disease research, implement this specialized methodology:

• Disease-specific application strategies:

  • Alzheimer's disease: Examine PERK activation in relation to Aβ plaques and tau tangles

  • Progressive supranuclear palsy: Investigate PERK in the context of tau pathology

  • Parkinson's disease: Study PERK activation in α-synuclein models

  • ALS: Assess PERK in relation to TDP-43 and C9orf72 pathology

• Genetic risk assessment:

  • Genotype samples for EIF2AK3 variants (rs867529, rs13045, rs1805165)

  • Stratify analyses based on PERK-A vs. PERK-B haplotypes

  • Examine gene-gene interactions (particularly with APOE genotype)

• Cell-type specific analysis:

  • Implement multiplexed immunofluorescence with cell-type markers

  • Use single-cell or nuclei isolation techniques followed by immunoblotting

  • Apply cell sorting prior to biochemical analysis

• Spatial distribution characterization:

  • Map PERK activation patterns in relation to disease pathology

  • Compare affected vs. spared brain regions

  • Assess subcellular localization changes during disease progression

• Therapeutic target validation:

  • Test PERK modulators in disease models

  • Evaluate downstream pathway interventions

  • Assess combination approaches targeting multiple UPR branches

• Biomarker development:

  • Correlate tissue PERK activation with fluid biomarkers

  • Assess phospho-PERK levels in accessible patient samples

  • Develop surrogate markers for PERK pathway activation

This comprehensive approach leverages EIF2AK3 antibodies to address critical questions in neurodegeneration research .

What methodological approaches should be used when studying EIF2AK3 in diabetes and pancreatic β-cell research?

When studying EIF2AK3 in diabetes and pancreatic β-cell research, implement these methodological approaches:

• Islet-specific techniques:

  • Optimize immunostaining protocols for pancreatic sections (different fixation methods)

  • Develop islet isolation procedures that preserve PERK phosphorylation status

  • Implement live imaging of pancreatic slices with fluorescent PERK activity reporters

• Diabetes model characterization:

  • Compare PERK activation across type 1 and type 2 diabetes models

  • Analyze Wolcott-Rallison syndrome (WRS) models with PERK mutations

  • Assess diet-induced vs. genetic models of β-cell stress

• β-cell stress-response analysis:

  • Measure temporal dynamics of PERK activation during stress exposure

  • Correlate PERK activity with β-cell function (glucose-stimulated insulin secretion)

  • Assess PERK's role in β-cell dedifferentiation vs. apoptosis

• Genetic manipulation approaches:

  • Implement β-cell-specific PERK knockout models

  • Generate knock-in models with human EIF2AK3 variants

  • Develop inducible systems for temporal control of PERK modulation

• Therapeutic intervention strategies:

  • Test chemical chaperones to alleviate ER stress

  • Evaluate PERK inhibitors/modulators for β-cell protection

  • Assess combination approaches targeting multiple stress pathways

• Translational applications:

  • Analyze PERK activation in human pancreatic samples

  • Correlate genotype (EIF2AK3 variants) with islet phenotypes

  • Develop biomarkers reflecting β-cell PERK activation state

This comprehensive methodology enables robust investigation of PERK's role in pancreatic β-cell biology and diabetes pathogenesis .

How can researchers optimize the use of EIF2AK3 antibodies in cancer research studies?

To optimize the use of EIF2AK3 antibodies in cancer research studies, implement this specialized methodology:

• Tumor-specific application protocols:

  • Optimize tissue processing to preserve phosphorylation status in clinical samples

  • Develop tissue microarray (TMA) approaches for high-throughput screening

  • Implement multiplexed detection with cancer-specific markers

• Cancer progression analysis:

  • Compare PERK activation across tumor stages/grades

  • Assess correlation with metastatic potential

  • Evaluate changes during treatment response and resistance development

• Microenvironment interaction studies:

  • Examine PERK activation in hypoxic tumor regions

  • Assess nutrient deprivation effects on PERK signaling

  • Investigate tumor-stroma interactions through dual staining approaches

• Therapeutic resistance mechanisms:

  • Correlate PERK activation with drug resistance phenotypes

  • Examine PERK-dependent adaptive responses to targeted therapies

  • Develop combination approaches targeting UPR alongside conventional treatments

• Patient stratification strategies:

  • Develop immunohistochemical scoring systems for PERK activation

  • Correlate PERK activity with clinical outcomes

  • Assess EIF2AK3 genetic variants as predictive/prognostic markers

• Functional validation approaches:

  • Implement CRISPR-based PERK modulation in patient-derived xenografts

  • Develop organoid models with altered PERK signaling

  • Utilize in vivo imaging of PERK activity in tumor models

This comprehensive methodology enables researchers to leverage EIF2AK3 antibodies for significant advances in cancer biology and therapeutic development .