EIF2AK2 Antibody, HRP conjugated

What is EIF2AK2 and why is it important for research involving HRP-conjugated antibodies?

EIF2AK2, also known as PKR (Protein Kinase R), is a serine/threonine protein kinase activated by autophosphorylation after binding to double-stranded RNA. This 62 kDa protein (calculated MW) typically appears as 65-74 kDa bands in Western blots due to post-translational modifications .

EIF2AK2 functions by phosphorylating translation initiation factor EIF2S1, inhibiting protein synthesis in response to various cellular stresses. Its biological significance spans multiple areas:

• Viral infection response and innate immunity regulation

• Cancer biology (both tumor-suppressive and oncogenic roles)

• Neurological disorders (implicated in early-onset dystonia)

• Cellular stress response mechanisms

For research applications, HRP-conjugated EIF2AK2 antibodies offer direct enzymatic detection without secondary antibodies, enabling more consistent results when analyzing expression patterns across different experimental conditions.

How do I select the appropriate HRP-conjugated EIF2AK2 antibody for my specific application?

Selection should be methodologically driven by your experimental goals:

Methodological approach:

• Examine validation data (specificity verified with knockout cells is ideal)

• Review published applications in similar experimental systems

• Test multiple antibodies targeting different epitopes when possible

• For phosphorylation studies, select antibodies specifically validated for distinguishing activation states

What are the most effective protocols for sample preparation when using HRP-conjugated EIF2AK2 antibodies?

Sample preparation significantly impacts antibody performance. The following methodological approaches optimize detection:

For Western Blot samples:

• Cell lysis buffer selection:

  • RIPA buffer with phosphatase inhibitors preserves phosphorylation status

  • Add protease inhibitors to prevent degradation (particularly important for EIF2AK2)

  • Include 1-5 mM DTT as EIF2AK2 contains multiple cysteine residues

• Protein quantification and loading:

  • Load 20-25 μg of total protein per lane (based on protocols from multiple sources)

  • Use reducing conditions with SDS-PAGE for optimal separation

  • Expected MW: 65-74 kDa (higher than calculated 62 kDa due to phosphorylation)

• SDS-PAGE conditions:

  • 8-10% gels provide optimal resolution around 62-74 kDa

  • Complete transfer to membrane at 30V overnight (or 100V for 1 hour) for proteins >60 kDa

For Immunohistochemistry:

• Tissue preparation:

  • Antigen retrieval with TE buffer (pH 9.0) is recommended based on validation data

  • Alternatively, citrate buffer (pH 6.0) has been used successfully

  • Block endogenous peroxidase with 3% hydrogen peroxide before primary antibody

• Staining controls:

  • Include known positive tissue samples (e.g., pancreatic cancer tissue)

  • Use adjacent normal tissue as relative expression control

How do I interpret variable EIF2AK2 molecular weights observed in Western blots?

EIF2AK2 frequently shows discrepancies between calculated (62 kDa) and observed (65-74 kDa) molecular weights. Understanding these variations is critical for accurate interpretation:

Molecular weight interpretation guide:

Methodologically, verify these interpretations by:

• Treating lysates with lambda phosphatase to eliminate phosphorylation-dependent bands

• Stimulating cells with IFN-α or poly(I:C) to enrich activated forms

• Including EIF2AK2 knockout samples as negative controls

• Running gradient gels for better resolution of closely migrating forms

How can I use HRP-conjugated EIF2AK2 antibodies to investigate its role in cancer biology?

EIF2AK2 exhibits complex, context-dependent roles in cancer. Recent research shows it may function as either tumor suppressor or oncogene depending on cancer type . For pancreatic cancer specifically, EIF2AK2 expression is significantly higher in tumor tissues than adjacent normal tissue and correlates with immune infiltration .

Methodological approach for cancer studies:

• Expression analysis across cancer types:

  • Compare total EIF2AK2 levels between tumor and matched normal tissues

  • Analyze publicly available datasets (TCGA, GTEx, GEO) as reference points

  • Consider datasets GSE15471, GSE16515, GSE32676, and GSE62165 for pancreatic cancer

• Correlation with clinical outcomes:

  • Stratify patients by EIF2AK2 expression level (high vs. low)

  • Perform Kaplan-Meier analysis to assess survival correlations

  • Use Cox regression to evaluate prognostic significance

• Immune infiltration analysis:

  • Investigate correlations with immune cell populations using ssGSEA

  • Analyze relationship with immune checkpoints:

  Immune Checkpoint	Correlation with EIF2AK2	Statistical Significance
  CD274 (PD-L1)	Strong positive (rs=0.601)	P<0.001
  PDCD1LG2	Strong positive (rs=0.501)	P<0.001
  HAVCR2	Moderate positive (rs=0.423)	P<0.001
  TIGIT	Moderate positive (rs=0.341)	P<0.001
  CTLA4	Moderate positive (rs=0.311)	P<0.001
  PDCD1	Weak positive (rs=0.221)	P=0.003
  LAG3	Weak positive (rs=0.207)	P=0.006

• Functional validation:

  • Knockdown/knockout EIF2AK2 in cancer cell lines

  • Evaluate effects on proliferation, migration, and response to therapy

  • Analyze changes in immune checkpoint expression after EIF2AK2 modulation

How do phosphorylation-specific EIF2AK2 antibodies help in understanding its activation mechanism?

EIF2AK2 activation involves a complex series of phosphorylation events. Recent research has identified novel regulatory phosphorylation sites that significantly impact activity .

Key phosphorylation sites and their functions:

• Primary activation sites:

  • Thr446 and Thr451: Critical for kinase activation following dimerization

  • Phospho-specific antibodies targeting these sites indicate active EIF2AK2

• Newly discovered regulatory sites:

  • Ser6: Located 3 amino acids upstream of DRBM1

  • Ser97: Located in same position relative to DRBM2

• Regulatory mechanisms:

  Phosphorylation Site	Effect When Phosphorylated	Effect When Mutated to Alanine
  Ser6/Ser97	Maintains inactive conformation	Spontaneous activation
  Thr446/Thr451	Required for kinase activity	Prevents activation

Methodological approach with phospho-specific antibodies:

• Activation state assessment:

  • Use phospho-Thr446/Thr451 antibodies to quantify active EIF2AK2

  • Normalize to total EIF2AK2 to calculate activation percentage

• Regulatory phosphorylation analysis:

  • Develop antibodies targeting newly identified Ser6/Ser97 sites

  • Monitor changes in regulatory phosphorylation under different stimuli

• Mutational analysis validation:

  • Generate phosphomimetic (S→D) and phospho-dead (S→A) mutants

  • Compare antibody reactivity with wild-type and mutant proteins

  • Correlate phosphorylation patterns with functional outcomes

When interpreting results, remember that negative charges at positions 6 and 97 appear to tighten interactions between RNA-binding motifs and kinase domain, maintaining EIF2AK2 in an inactive conformation even in the presence of dsRNA .

How can I use EIF2AK2 antibodies to investigate immune responses in viral infections?

EIF2AK2 plays a critical role in antiviral immunity, making it an important target for infectious disease research. The protein is activated by viral dsRNA and inhibits viral protein synthesis through eIF2α phosphorylation.

Methodological approach for viral infection studies:

• Expression and activation monitoring:

  • Track total EIF2AK2 levels during infection time course

  • Monitor phosphorylation status using phospho-specific antibodies

  • Correlate with viral replication markers

• Intervention studies:

  • Compare wild-type vs. EIF2AK2-deficient cells

  • Assess viral replication efficiency

  • Measure interferon response pathways

• Interferon stimulated gene (ISG) analysis:
  Recent findings show EIF2AK2 regulates ISG expression through mRNA splicing :

  ISG	Regulation by EIF2AK2	Verification Method
  Mx1	Positive regulation through splicing	qRT-PCR of specific variants
  OAS1	Positive regulation through splicing	Western blot protein detection
  PKR (auto-regulation)	Self-regulation through splicing	Expression rescue experiments

• Mechanistic investigations:

  • EIF2AK2 single allele knockout reduces ISG protein expression

  • Spliceosome factor EFTUD2 mediates IFN anti-HBV effects through regulation of ISG splicing, including EIF2AK2

  • Rescue experiments through EIF2AK2 overexpression can confirm specificity

When designing experiments, consider that IFN-α treatment increases EIF2AK2 expression, making it a useful positive control for antibody validation .

What methodological approaches can investigate the role of EIF2AK2 in neurological disorders?

Recent discoveries have linked EIF2AK2 missense variants with early onset generalized dystonia (DYT33) , opening new research directions in neurological disorders.

Key research strategies:

• Genetic variant analysis:

  • Screen for EIF2AK2 variants in dystonia patients

  • Focus on variants identified in previous studies:

    • c.388G>A (p.Gly130Arg): Found in multiple families, including a de novo case

    • c.413G>C (p.Gly138Ala): Different heterozygous variant

    • c.95A>C (p.Asn32Thr): Homozygous variant in consanguineous family

• Functional characterization:

  • Express variant proteins in cellular models

  • Compare phosphorylation patterns with wild-type EIF2AK2

  • Assess kinase activity toward eIF2α

• Splicing analysis:

  • Examine potential splicing effects of variants near exon-intron boundaries

  • Perform cDNA studies to detect aberrant splicing products

  • Use minigene assays to confirm variant effects on splicing

• Neuronal model systems:

  • Develop neuronal models expressing EIF2AK2 variants

  • Assess impact on protein synthesis regulation

  • Investigate effects on neuronal morphology and function

When using HRP-conjugated antibodies in these studies, ensure they can detect the variant proteins equally well as wild-type, possibly by targeting conserved epitopes away from the mutation sites.

What are the most effective strategies for troubleshooting weak or absent EIF2AK2 signal?

When facing detection challenges with EIF2AK2 antibodies, a systematic troubleshooting approach is essential:

Signal enhancement methods:

• Sample preparation optimization:

  • Increase protein loading (up to 25-30 μg per lane)

  • Add phosphatase inhibitors to preserve phosphorylated forms

  • Use fresh lysates to minimize degradation

• Antibody conditions adjustment:

  • Reduce antibody dilution (start with 1:500 for weak signals)

  • Extend incubation time (overnight at 4°C instead of 1-2 hours at RT)

  • Switch to more sensitive detection substrates (enhanced ECL)

• Signal amplification techniques:

  • For HRP-conjugated antibodies: Use high-sensitivity substrates with longer exposure times

  • Consider tyramide signal amplification for IHC/IF applications

  • Try reduced-size format blots to concentrate protein

• Expression enhancement:

  • Treat cells with IFN-α to upregulate EIF2AK2 expression

  • Use cell lines known to express higher EIF2AK2 levels (HeLa, HEK293, K562)

  • Consider transfection with EIF2AK2 expression constructs as positive controls

Decision matrix for troubleshooting weak signals:

How can I optimize Western blot protocols specifically for HRP-conjugated EIF2AK2 antibodies?

HRP-conjugated antibodies require specific optimization strategies for maximum sensitivity and specificity:

Optimized Western blot protocol:

• Membrane preparation:

  • After transfer, rinse membrane in TBS to remove methanol

  • Block in 3-5% nonfat dry milk in TBST for 1 hour at room temperature

  • For phospho-specific detection, use 5% BSA instead of milk

• Antibody incubation:

  • Dilute HRP-conjugated EIF2AK2 antibody in fresh blocking buffer

  • Start with manufacturer's recommended dilution (typically 1:1000-1:2000)

  • Incubate 1 hour at room temperature or overnight at 4°C with gentle agitation

  • Avoid sodium azide in antibody dilution buffer as it inhibits HRP

• Washing optimization:

  • Perform 5-6 washes with TBST, 5 minutes each

  • Increase wash volume to at least 4 mL per cm² of membrane

  • Use fresh wash buffer for each wash

• Detection conditions:

  • Use freshly prepared ECL substrate

  • For weak signals, extend substrate incubation to 2-5 minutes

  • Start with 30-second exposure and adjust as needed

  • For quantitative analysis, capture multiple exposures to ensure linear range

Validation controls:

• Include wild-type and EIF2AK2 knockout cell lysates (e.g., A549 or HeLa KO lines)

• Use GAPDH (36 kDa) as loading control to verify equal protein loading

What are the critical factors in validating new HRP-conjugated EIF2AK2 antibodies for research applications?

Thorough validation is essential before using any new EIF2AK2 antibody for research applications. The following methodological approach ensures reliable results:

Comprehensive validation strategy:

• Specificity verification:

  • Test against EIF2AK2 knockout cell lysates (gold standard)

  • Compare with existing validated antibodies targeting different epitopes

  • Perform immunoprecipitation followed by mass spectrometry to confirm target

• Functional validation:

  • Verify appropriate response to stimuli (e.g., increased phosphorylation after dsRNA treatment)

  • Check for expected subcellular localization patterns

  • Confirm expected molecular weight (typically 65-74 kDa for EIF2AK2)

• Application-specific testing:

  Application	Validation Method	Success Criteria
  Western Blot	Serial dilution	Linear response across concentration range
  	Multiple cell lines	Detection in HeLa, HEK293, K562, Jurkat, MCF-7
  IHC	Known positive tissues	Specific staining in testis, kidney tissue
  	Signal controls	Signal elimination with blocking peptide
  IP	MS confirmation	EIF2AK2 as top identified protein

• HRP conjugation-specific tests:

  • Verify HRP activity with direct substrate test

  • Compare signal-to-noise ratio with unconjugated primary + HRP-secondary approach

  • Assess stability over time with repeated testing of the same antibody lot

• Literature cross-validation:

  • Compare your results with published patterns of EIF2AK2 expression/activation

  • Reference known molecular weights from previous studies (65-74 kDa range)

How do post-translational modifications affect EIF2AK2 detection, and how should I account for these in my experiments?

EIF2AK2 undergoes multiple post-translational modifications (PTMs) that significantly impact its detection:

Major PTMs affecting detection:

• Phosphorylation:

  • Primary activation: Thr446 and Thr451

  • Regulatory sites: Ser6 and Ser97 recently identified

  • Effect on detection: Increases apparent molecular weight by 2-8 kDa

• Other potential modifications:

  • Ubiquitination: May create higher MW bands

  • SUMOylation: Can alter protein migration

  • Acetylation: May affect antibody binding to specific epitopes

Experimental strategies to address PTM-related issues:

• PTM-specific analysis:

  • Use phospho-specific antibodies to detect activation status

  • Employ phosphatase treatment to confirm phosphorylation-dependent bands

  • Run samples under conditions that preserve or remove specific modifications

• Protocol adjustments:

  PTM Consideration	Methodological Adjustment	Rationale
  Phosphorylation	Add phosphatase inhibitors to lysis buffer	Preserves phosphorylation status
  	Include phosphorylated controls	Positive control for activation
  Degradation	Add protease inhibitors	Prevents protein degradation
  Multiple forms	Use gradient gels (4-15%)	Better separation of different forms
  Activation analysis	Compare resting vs. stimulated cells	Demonstrates dynamic changes

• Visualization strategies:

  • For total EIF2AK2: Use antibodies targeting conserved regions away from PTM sites

  • For activation studies: Compare phospho-specific to total EIF2AK2 signal

  • For PTM mapping: Consider 2D gel electrophoresis followed by Western blot

• Stimulation protocols for validation:

  • dsRNA mimics (poly(I:C)): Directly activates EIF2AK2

  • IFN-α treatment: Increases expression and sensitizes to activation

  • Viral infection: Natural activator of the PKR pathway

How should I design experiments to investigate EIF2AK2's role in regulating gene expression through RNA splicing?

Recent research has revealed EIF2AK2's involvement in regulating gene expression through mRNA splicing, particularly for interferon-stimulated genes (ISGs) . This opens new research directions requiring specialized experimental approaches:

Comprehensive experimental design:

• Gene expression analysis:

  • Compare transcriptomes between wild-type and EIF2AK2-deficient cells

  • Analyze RNA-seq data with splice-aware alignment tools

  • Focus on known ISGs (Mx1, OAS1) affected by EIF2AK2

• Splicing event characterization:

  • Perform RT-PCR with primers spanning potential splice junctions

  • Use exon-specific primers to quantify individual splicing events

  • Design primers for specific transcript variants identified in RNA-seq

• Functional validation:

  Approach	Methodology	Expected Outcome
  Rescue experiments	Reintroduce EIF2AK2 into knockout cells	Restoration of normal splicing patterns
  Mutation analysis	Express kinase-dead EIF2AK2	Determine if kinase activity is required for splicing
  Domain mapping	Express truncated EIF2AK2 constructs	Identify domains critical for splicing regulation

• Interaction studies with splicing machinery:

  • Investigate interaction with EFTUD2 (spliceosome factor)

  • Perform co-immunoprecipitation with spliceosome components

  • Use RNA immunoprecipitation to identify directly bound transcripts

When using HRP-conjugated EIF2AK2 antibodies in these studies, ensure specificity by including appropriate knockout controls and comparing results with unconjugated antibodies to rule out any HRP-related artifacts.

How can multiplexed immunoassays with EIF2AK2 antibodies provide insights into pathway activation?

Multiplexed detection allows simultaneous analysis of EIF2AK2 and related proteins, providing comprehensive pathway insights:

Multiplexed analysis strategies:

• Multi-color Western blotting:

  • Combine HRP-conjugated EIF2AK2 antibody with differently labeled antibodies

  • Detect total and phospho-EIF2AK2 simultaneously

  • Include downstream markers (p-eIF2α, ATF4, CHOP)

• Pathway activation profiling:

  Target	Significance	Expected Pattern in Activation
  EIF2AK2 (total)	Expression level	Increased after IFN treatment
  p-EIF2AK2 (T446/T451)	Activation marker	Rapidly increased after dsRNA exposure
  p-eIF2α (S51)	Downstream effect	Follows p-EIF2AK2 with slight delay
  ATF4	Integrated stress response	Increased translation despite global inhibition
  CHOP	Terminal stress response	Increased in prolonged activation

• Multi-parameter flow cytometry:

  • Permeabilize cells to detect intracellular EIF2AK2

  • Combine with surface markers to identify responsive cell populations

  • Include viability markers to distinguish stress responses

• Tissue microarray analysis:

  • Compare EIF2AK2 levels across multiple samples simultaneously

  • Correlate with phospho-EIF2AK2 and downstream markers

  • Analyze relationship with immune cell markers based on correlation with immune infiltration

When designing multiplexed assays with HRP-conjugated antibodies, careful planning is needed to avoid signal overlap. Consider sequential detection protocols or use HRP inactivation between detection steps.

What approaches can effectively measure EIF2AK2 activation dynamics in live cell imaging experiments?

Studying EIF2AK2 activation dynamics requires specialized techniques to capture real-time changes:

Live cell imaging methodologies:

• Fluorescent reporter systems:

  • Generate EIF2AK2-fluorescent protein fusion constructs

  • Create phospho-specific biosensors based on FRET technology

  • Use destabilized fluorescent proteins under control of ATF4 regulatory elements

• Activation dynamics monitoring:

  Phenomenon	Measurement Approach	Expected Timeline
  Dimerization	FRET or BiFC between tagged EIF2AK2 molecules	Minutes after dsRNA exposure
  Phosphorylation	Phospho-specific biosensors	15-30 minutes after activation
  Translational inhibition	Fluorescent translation reporters	30-60 minutes after activation
  Stress granule formation	Co-localization with G3BP1 markers	1-2 hours after sustained activation

• Advanced microscopy techniques:

  • Use confocal microscopy to track subcellular localization changes

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to measure mobility changes upon activation

  • Implement light-sheet microscopy for 3D visualization of activation patterns

• Quantitative analysis methods:

  • Track intensity changes in specific cellular compartments

  • Measure nuclear-cytoplasmic ratios over time

  • Quantify stress granule formation and composition

While HRP-conjugated antibodies aren't directly applicable to live cell imaging, fixed-cell validation experiments using these antibodies can confirm the specificity of fluorescent tags and biosensors.

How should I interpret seemingly contradictory findings about EIF2AK2's role in different disease contexts?

The search results reveal apparently contradictory roles for EIF2AK2 across different diseases and cellular contexts . Resolving these contradictions requires careful methodological consideration:

Framework for reconciling contradictory findings:

• Context-dependent function analysis:

  • Cancer context: Both tumor suppressive and oncogenic roles reported

  • Neurological context: Associated with early-onset dystonia

  • Viral infection: Generally protective through translation inhibition

• Tissue-specific effects:

  Tissue/Cancer Type	Reported EIF2AK2 Function	Suggested Mechanism
  Breast, lung, colorectal	Tumor suppressive	Loss associated with poor prognosis
  Pancreatic cancer	Potentially oncogenic	Higher expression in tumors vs. normal tissue
  Hepatocellular carcinoma	Proliferation/migration promoter	Mechanism not fully characterized
  Gastric cancer	Metastasis promoter	Mechanism not fully characterized
  Neuronal tissue	Pathogenic when mutated	Missense variants linked to dystonia

• Pathway interaction considerations:

  • Immune context: EIF2AK2 correlates with immune checkpoint molecules (CD274, PDCD1LG2)

  • Splicing regulation: Interactions with EFTUD2 affect ISG expression

  • Phosphorylation sites: Different sites have opposing effects on activity

• Experimental approach harmonization:

  • Use consistent cell models across studies

  • Apply multiple methodologies to verify findings

  • Consider kinetics and dose-response relationships

  • Distinguish between correlation and causation through mechanistic studies

When conducting your own research on EIF2AK2, clearly define the cellular context, document all experimental conditions, and employ multiple complementary methods to build confidence in your findings.

What reference materials and controls are essential for EIF2AK2 antibody experiments?

Robust EIF2AK2 research requires carefully selected controls and reference materials:

Essential reference materials:

• Cell line controls:

  • Positive expression controls: HeLa, HEK-293, K562, Jurkat, MCF-7 cells

  • Negative controls: EIF2AK2 knockout cell lines (A549, HeLa)

  • Expression enhancement: IFN-α treated cells show increased EIF2AK2 levels

• Activation controls:

  Control Type	Purpose	Implementation
  Basal state	Baseline expression	Serum-starved, unstimulated cells
  Activated state	Positive control for phosphorylation	Poly(I:C) transfected cells
  Inhibited state	Negative control for activation	PKR inhibitor (C16) treated cells
  Kinase-dead control	Specificity control	K296R mutant expression

• Technical controls:

  • Loading control: GAPDH (36 kDa) works well with EIF2AK2 detection

  • HRP activity control: Test substrate directly on membrane

  • Antibody specificity: Pre-incubation with immunizing peptide

• Reference datasets:

  • Pancreatic cancer expression: GSE15471, GSE16515, GSE32676, GSE62165

  • Normal tissue expression: GTEx database (167 non-malignant pancreas samples)

  • Cancer expression: TCGA-PAAD dataset (178 tumor samples)

How can I optimize EIF2AK2 antibody protocols for different tissue types and preparations?

Different tissue types require specific optimization strategies for reliable EIF2AK2 detection:

Tissue-specific optimization guidelines:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Antigen retrieval: TE buffer pH 9.0 recommended for EIF2AK2

  • Alternative: Citrate buffer pH 6.0 has also been successful

  • Section thickness: 4-5 μm optimal for antibody penetration

  • Antibody dilution: Start with 1:50-1:100 for IHC applications

• Frozen tissue sections:

  • Fixation: 4% paraformaldehyde for 10 minutes

  • Permeabilization: 0.1-0.5% Triton X-100 for intracellular access

  • Blocking: 1-2 hours with 5-10% normal serum

  • Higher antibody dilutions (1:200-1:500) may be effective

• Tissue-specific considerations:

  Tissue Type	Special Considerations	Protocol Adjustments
  Pancreatic tissue	High endogenous peroxidase	Extended peroxidase blocking (15 min)
  	High background with milk	Use BSA or commercial blockers
  Brain tissue	Lipid-rich environment	Add 0.1% Tween-20 to antibody diluent
  	Autofluorescence	Treat with Sudan Black B before antibody
  Tumor tissues	Variable expression	Include internal controls within section
  	Heterogeneous expression	Analyze multiple fields (≥5)

• Tissue microarrays (TMAs):

  • Single standardized protocol applies to all samples

  • Include known positive and negative controls in each TMA

  • Score intensity using standardized scales (0-3+)

  • Consider digital image analysis for quantification

For all tissue preparations, validate staining patterns with multiple antibodies and correlate with mRNA expression data when available.

What are the most effective methods for quantifying EIF2AK2 expression and activation levels?

Accurate quantification is essential for meaningful EIF2AK2 research. Multiple approaches offer complementary advantages:

Comprehensive quantification methods:

• Western blot quantification:

  • Capture images within linear dynamic range

  • Use lane profile analysis in software like ImageJ

  • Normalize to loading controls (GAPDH recommended)

  • Express as fold-change relative to control conditions

• Densitometric analysis guidelines:

  Measurement	Calculation Method	Application
  Total EIF2AK2	EIF2AK2 band intensity / GAPDH intensity	Expression level changes
  Activation ratio	Phospho-EIF2AK2 / Total EIF2AK2	Activation state analysis
  Relative expression	Sample intensity / Reference sample intensity	Cross-comparison between conditions

• Flow cytometry quantification:

  • Use geometric mean fluorescence intensity (gMFI)

  • Include isotype controls to set negative gates

  • Calculate stain index: (Sample MFI - Background MFI) / 2× SD of background

  • Compare with standard curves for absolute quantification

• Real-time PCR correlation:

  • Primer design for specific EIF2AK2 transcript variants

  • Normalize to validated reference genes

  • Calculate relative expression using 2^(-ΔΔCt) method

  • Correlate mRNA with protein levels to assess post-transcriptional regulation

• Digital pathology approaches:

  • Whole slide scanning of immunohistochemistry

  • Automated detection of positive cells

  • Intensity scoring (0, 1+, 2+, 3+)

  • Both percentage of positive cells and intensity should be reported

When quantifying HRP-conjugated antibody signals, ensure substrate isn't depleted (non-linear range) by testing multiple exposure times or dilution series.