Phospho-EIF2S1 (S51) Recombinant Monoclonal Antibody

What is EIF2S1 and what role does phosphorylation at S51 play in cellular function?

EIF2S1 (eIF2-alpha) is a key subunit of the eukaryotic translation initiation factor 2 (eIF2) complex that functions in the early steps of protein synthesis. This complex forms a ternary complex with GTP and initiator tRNA, which then binds to a 40S ribosomal subunit followed by mRNA binding to form a 43S pre-initiation complex (43S PIC) . Phosphorylation at serine 51 (S51) transforms EIF2S1 into a global protein synthesis inhibitor, as this modification prevents the recycling of the eIF2 complex by inhibiting the guanine nucleotide exchange factor eIF2B .

The phosphorylation of EIF2S1 at S51 is mediated by four dedicated stress-sensing kinases: EIF2AK1/HRI (responding to heme deficiency and oxidative stress), EIF2AK2/PKR (activated by viral infection), EIF2AK3/PERK (triggered by endoplasmic reticulum stress), and EIF2AK4/GCN2 (responding to amino acid deficiency) . This phosphorylation event represents a central node in the integrated stress response (ISR) pathway, allowing cells to rapidly adapt to various stressors by reprogramming translation and gene expression.

How does phospho-EIF2S1 (S51) contribute to transcriptional reprogramming during stress?

When EIF2S1 becomes phosphorylated at S51 during cellular stress, it leads to a dual effect on translation: general inhibition of cap-dependent translation while simultaneously promoting the preferential translation of specific stress-responsive mRNAs . This selective translation includes transcriptional activators such as ATF4 and QRICH1, which orchestrate expression of genes involved in stress adaptation .

The stress-induced translation of ATF4 and other transcription factors leads to the activation of downstream genes involved in amino acid metabolism, redox homeostasis, autophagy, and cell fate decisions. For example, studies with EIF2S1 S51A mutant (phosphorylation-deficient) cells have demonstrated defects in autophagosome and autolysosome formation under ER stress conditions, highlighting the essential role of phospho-EIF2S1 in autophagy regulation .

Notably, phosphorylated EIF2S1 facilitates the nuclear translocation of transcription factors TFEB and TFE3, which are master regulators of autophagy and lysosomal biogenesis. This process requires cooperation with other unfolded protein response (UPR) components, including EIF2AK3/PERK and PPP3/calcineurin-mediated dephosphorylation mechanisms .

What experimental applications are most suitable for phospho-EIF2S1 (S51) recombinant monoclonal antibodies?

Phospho-EIF2S1 (S51) recombinant monoclonal antibodies have been validated for multiple experimental applications with varying levels of reliability:

The recombinant format of these antibodies, particularly the E90 clone, provides exceptional batch-to-batch consistency, eliminating the need for same-lot requests that often plague research utilizing polyclonal antibodies . This consistency is crucial for longitudinal studies and comparing results across different experimental timepoints.

Which model systems have been validated for phospho-EIF2S1 (S51) antibody applications?

Phospho-EIF2S1 (S51) recombinant monoclonal antibodies have been validated across multiple model systems:

When using these antibodies in novel model systems, researchers should conduct preliminary validation experiments due to potential species-specific differences in epitope accessibility or post-translational modification patterns surrounding the phosphorylation site .

How can phospho-EIF2S1 (S51) antibodies be used to investigate different branches of the integrated stress response?

Phospho-EIF2S1 (S51) antibodies serve as essential tools for dissecting the activation of different integrated stress response (ISR) branches. Each of the four EIF2S1 kinases (EIF2AK1-4) responds to distinct stress stimuli, and careful experimental design using phospho-specific antibodies can reveal which pathway is predominantly activated under specific conditions .

For systematic investigation of ISR branches, researchers can employ the following approach:

• Use selective stressors that preferentially activate specific kinases (e.g., thapsigargin for PERK activation, poly(I:C) for PKR activation)

• Compare phospho-EIF2S1 (S51) levels using recombinant monoclonal antibodies

• Validate pathway specificity using kinase-specific inhibitors or genetic knockdown/knockout models

• Examine downstream effectors like ATF4 and QRICH1 to confirm functional consequences

This methodological framework allows researchers to distinguish between primary stress responses and secondary adaptive mechanisms, providing insights into the complex interplay between different cellular stress pathways .

What are the optimal protocols for using phospho-EIF2S1 (S51) antibodies in multi-parameter stress response analysis?

When conducting multi-parameter analysis of stress responses using phospho-EIF2S1 (S51) antibodies, researchers should implement a comprehensive experimental design that captures both temporal dynamics and pathway interactions:

For optimal results, researchers should first perform a time-course analysis to determine the kinetics of EIF2S1 phosphorylation in their specific experimental system. This temporal profile will inform subsequent investigations of downstream effectors and functional outcomes .

How do phospho-EIF2S1 (S51) levels correlate with autophagy induction and progression?

Phosphorylation of EIF2S1 at S51 plays a critical role in the regulation of autophagy, particularly during endoplasmic reticulum stress. Research using EIF2S1 S51A mutant (A/A) cells has revealed that phospho-EIF2S1 is required for multiple steps of the autophagic process :

• Autophagosome formation: Phospho-EIF2S1 (S51) facilitates the nuclear translocation of TFEB and TFE3, master transcription factors controlling autophagy gene expression

• Autolysosome formation: The phosphorylation state of EIF2S1 influences fusion events between autophagosomes and lysosomes

• Transcriptional regulation: Downstream effectors of phospho-EIF2S1, such as ATF4, XBP1s, and ATF6, differentially contribute to autophagy gene expression

For accurate correlation analysis, researchers should monitor phospho-EIF2S1 (S51) levels alongside established autophagy markers such as LC3-II/LC3-I ratio, SQSTM1/p62 degradation, and autophagosome formation using fluorescence microscopy. Importantly, while EIF2S1 phosphorylation is necessary for stress-induced autophagy, it operates in conjunction with other regulatory mechanisms like PPP3/calcineurin-mediated dephosphorylation of TFEB/TFE3 .

What are the technical considerations for assessing phospho-EIF2S1 (S51) in mitophagy studies?

Recent research has identified phospho-EIF2S1 (S51) as an activator of mitophagy in response to mitochondrial damage. Specifically, phosphorylation by EIF2AK1/HRI promotes relocalization of EIF2S1 to the mitochondrial surface, triggering PRKN-independent mitophagy . When investigating this process, researchers should consider the following technical aspects:

• Subcellular fractionation quality: Clean separation of mitochondrial fractions is essential for accurate assessment of EIF2S1 relocalization

• Temporal resolution: The kinetics of EIF2S1 phosphorylation and mitochondrial association may vary depending on the type and intensity of stress

• Dual immunostaining approach: Combine phospho-EIF2S1 (S51) antibodies with mitochondrial markers in immunofluorescence studies

• Quantification methods: Develop consistent parameters for assessing mitochondrial association of phospho-EIF2S1

Additionally, researchers should validate their findings using pharmacological modulators of the EIF2AK1/HRI pathway and genetic approaches such as EIF2S1 S51A mutants to establish causality between phosphorylation and mitophagy induction .

What are the validated protocols for using phospho-EIF2S1 (S51) antibodies in Western blot analysis?

For optimal detection of phospho-EIF2S1 (S51) by Western blot, researchers should follow these validated protocols:

• Sample preparation:

  • Harvest cells at optimal time points post-stress induction

  • Lyse cells in buffer containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Include protease inhibitors to prevent degradation

  • Process samples rapidly and maintain cold temperatures throughout

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Transfer to PVDF membranes (preferred over nitrocellulose for phospho-epitopes)

  • Verify transfer efficiency with Ponceau S staining

• Antibody incubation:

  • Block membranes in 5% BSA (not milk, which contains phosphatases)

  • Dilute phospho-EIF2S1 (S51) antibodies according to manufacturer recommendations (typically 1:1000-1:2000)

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

  • Use appropriate HRP-conjugated secondary antibodies

• Controls and normalization:

  • Include positive controls (cells treated with thapsigargin or other known inducers)

  • Use phosphorylation-deficient mutants (S51A) as negative controls when available

  • Strip and reprobe for total EIF2S1 for normalization

This protocol has been extensively validated with recombinant monoclonal antibodies such as the E90 clone, which has been trusted by researchers since 2006 and cited in over 270 publications .

How should samples be prepared for optimal detection of phospho-EIF2S1 (S51) in IHC-P applications?

For immunohistochemistry on paraffin-embedded sections (IHC-P), proper sample preparation is critical for preserving the phospho-epitope:

• Tissue fixation and preservation:

  • Fix tissues in 10% neutral-buffered formalin for 24-48 hours

  • Process tissues promptly to prevent phospho-epitope degradation

  • Consider using phosphatase inhibitors in fixation solutions

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is most effective

  • Maintain consistent retrieval times and temperatures across experimental groups

  • Optimize retrieval conditions for specific tissue types

• Blocking and antibody incubation:

  • Block with serum-free protein block to reduce background

  • Use validated antibody dilutions (typically 1:100-1:200 for IHC-P)

  • Extend primary antibody incubation time (overnight at 4°C) for improved sensitivity

• Detection and counterstaining:

  • Employ polymer-based detection systems for increased sensitivity

  • Calibrate DAB development time for optimal signal window

  • Use hematoxylin counterstain optimized for nuclear detail

These protocols have been validated using multi-tissue microarray (TMA) approaches to confirm both specificity and sensitivity across different tissue types . For phospho-EIF2S1 (S51) detection in stressed tissues, researchers should include appropriate control sections from unstressed samples for comparative analysis.

What controls are essential when working with phospho-EIF2S1 (S51) antibodies?

Rigorous controls are crucial for reliable interpretation of phospho-EIF2S1 (S51) data:

Additionally, researchers should implement dephosphorylation controls by treating duplicate samples with lambda phosphatase to confirm that the signal detected is indeed phosphorylation-dependent. For recombinant antibodies like clone E90, validation data from over 270 publications provides robust evidence for specificity across multiple applications .

How can phospho-EIF2S1 (S51) signal be quantified accurately across different experimental conditions?

Accurate quantification of phospho-EIF2S1 (S51) signals requires careful attention to both experimental design and data analysis:

• Experimental standardization:

  • Use consistent cell densities or tissue quantities across conditions

  • Process all experimental groups simultaneously

  • Include internal calibration standards when possible

  • Maintain identical exposure times for image acquisition

• Normalization strategies:

  • Calculate phospho-EIF2S1/total EIF2S1 ratio (preferred method)

  • Avoid normalizing to housekeeping proteins alone

  • Consider using total protein normalization methods (e.g., Stain-Free technology)

  • For IHC-P, quantify both staining intensity and percentage of positive cells

• Statistical analysis:

  • Perform multiple independent experiments (minimum n=3)

  • Use appropriate statistical tests based on data distribution

  • Account for multiple comparisons when analyzing complex experimental designs

  • Present both raw data and normalized results for transparency

• Validation approaches:

  • Correlate Western blot results with other methods (e.g., ELISA, phospho-flow cytometry)

  • Verify biological relevance by linking phosphorylation levels to downstream effects

By implementing these quantification protocols, researchers can achieve reliable comparative analysis of phospho-EIF2S1 (S51) levels across different experimental conditions, facilitating meaningful interpretation of integrated stress response activation .

How can researchers address common issues with phospho-EIF2S1 (S51) detection specificity?

Researchers commonly encounter several specificity issues when working with phospho-EIF2S1 (S51) antibodies:

Recombinant monoclonal antibodies, such as clone E90, offer superior specificity compared to polyclonal alternatives due to their defined epitope recognition. These antibodies undergo affinity purification using Protein A, further enhancing their specificity . When troubleshooting detection issues, researchers should first validate their experimental system using positive controls (e.g., thapsigargin-treated cells) to establish baseline expectations for signal intensity and pattern.

What approaches help resolve contradictory phospho-EIF2S1 (S51) data across different experimental models?

When faced with contradictory phospho-EIF2S1 (S51) data across different experimental models, researchers should implement a systematic resolution approach:

• Technical standardization:

  • Use identical antibody clones and protocols across all models

  • Standardize stress induction methods and timing

  • Process and analyze samples simultaneously

• Biological context assessment:

  • Evaluate baseline phosphatase activity in different models

  • Consider tissue-specific or cell type-specific regulation of EIF2S1 kinases

  • Assess expression levels of regulatory proteins (e.g., PPP1R15A/GADD34)

• Comprehensive pathway analysis:

  • Measure multiple components of the integrated stress response

  • Examine kinase activation upstream of EIF2S1

  • Evaluate functional outputs downstream of phosphorylation

• Validation with orthogonal methods:

  • Complement antibody-based detection with mass spectrometry

  • Use genetic approaches (e.g., phospho-mimetic mutants)

  • Correlate phosphorylation data with functional readouts

When documenting contradictory findings, researchers should clearly report experimental conditions, antibody information, and quantification methods to facilitate interpretation of discrepancies .

How should temporal dynamics of EIF2S1 phosphorylation be interpreted in stress response studies?

The temporal dynamics of EIF2S1 phosphorylation provide crucial insights into stress response mechanisms:

• Immediate response phase (0-30 minutes post-stress):

  • Rapid phosphorylation indicates direct kinase activation

  • The specific kinase involved can be inferred from the stressor type

  • This phase typically correlates with global translation inhibition

• Adaptive response phase (30 minutes - 4 hours):

  • Sustained phosphorylation reflects ongoing stress

  • Initiation of feedback mechanisms (e.g., GADD34 induction)

  • Correlation with ATF4 and other stress-responsive transcription factor levels

• Resolution phase (4-24 hours):

  • Declining phosphorylation indicates adaptation or recovery

  • Persistent phosphorylation may signal chronic stress or impaired homeostasis

  • Critical for distinguishing adaptive versus maladaptive responses

To accurately interpret these dynamics, researchers should:

• Conduct detailed time-course experiments with multiple timepoints

• Correlate phosphorylation patterns with functional outcomes

• Consider the potential influence of feedback mechanisms on phosphorylation levels

The phosphorylation of EIF2S1 at S51 represents a dynamic equilibrium between kinase and phosphatase activities, with implications for cell fate decisions during prolonged stress .

What are the best practices for normalizing phospho-EIF2S1 (S51) signals to total EIF2S1?

Accurate normalization of phospho-EIF2S1 (S51) signals to total EIF2S1 requires careful methodological consideration:

• Sequential detection approach:

  • Probe first for phospho-EIF2S1 (S51)

  • Document complete image data

  • Strip membranes thoroughly

  • Verify stripping efficiency

  • Reprobe for total EIF2S1

  • Use identical exposure parameters when possible

• Parallel detection method:

  • Run duplicate gels from the same samples

  • Transfer under identical conditions

  • Probe one membrane for phospho-EIF2S1 and one for total EIF2S1

  • Ensure equal loading with additional controls

• Quantification guidelines:

  • Use linear range of detection for both signals

  • Apply consistent analysis parameters

  • Calculate phospho/total ratio for each sample

  • Present both individual and ratio data

• Validation strategies:

  • Confirm consistent total EIF2S1 levels across conditions

  • Verify that stress does not alter total EIF2S1 expression

  • Include quality control samples across multiple blots

This rigorous normalization approach enables accurate assessment of the proportion of phosphorylated EIF2S1, which is the biologically relevant parameter in stress response studies .

How is phospho-EIF2S1 (S51) being utilized in studies of mitophagy?

Recent research has uncovered a novel role for phospho-EIF2S1 (S51) in the regulation of mitophagy—the selective autophagic degradation of damaged mitochondria. This connection represents an emerging frontier in cellular stress response research :

• Mechanistic insights:

  • Phosphorylation of EIF2S1 by EIF2AK1/HRI promotes its relocalization to the mitochondrial surface

  • This relocalization triggers PRKN-independent mitophagy

  • The process provides a direct link between translational control and mitochondrial quality control

• Experimental approaches:

  • Combination of subcellular fractionation with phospho-EIF2S1 (S51) immunoblotting

  • Co-localization studies using confocal microscopy

  • Functional assessment of mitophagy using mitochondrial reporters

  • Genetic manipulation using EIF2S1 S51A mutants

• Physiological significance:

  • Represents a novel pathway for maintaining mitochondrial homeostasis during cellular stress

  • May contribute to neuronal survival in neurodegenerative disorders

  • Potential therapeutic target for enhancing mitochondrial quality control

This research direction highlights the multifaceted roles of phospho-EIF2S1 beyond translational regulation and connects the integrated stress response to organelle-specific quality control mechanisms .

What is the emerging role of phospho-EIF2S1 (S51) in transcription factor regulation?

Phospho-EIF2S1 (S51) plays a previously underappreciated role in regulating transcription factor localization and activity, particularly for master regulators of autophagy and lysosomal biogenesis :

• TFEB and TFE3 regulation:

  • EIF2S1 phosphorylation is required for nuclear translocation of TFEB and TFE3 during ER stress

  • This mechanism operates in conjunction with PPP3/calcineurin-mediated dephosphorylation

  • YWHA/14-3-3 protein dissociation is necessary but not sufficient for nuclear retention

• Coordination with UPR pathways:

  • Different UPR components (ATF6, XBP1s, ATF4) differentially rescue defects in TFEB/TFE3 nuclear translocation

  • Activated ATF6 and TFEB most efficiently rescue autophagic defects in EIF2S1 phosphorylation-deficient cells

  • Reveals complex crosstalk between UPR branches and autophagy regulation

• Experimental approaches:

  • Analysis of nuclear/cytoplasmic fractionation in wild-type versus S51A mutant cells

  • Phosphorylation site mapping of TFEB/TFE3

  • Assessment of transcription factor binding to target promoters

  • Evaluation of autophagy gene expression profiles

This emerging research area demonstrates how phospho-EIF2S1 (S51) serves as a critical node connecting stress sensing, translational reprogramming, and transcriptional responses to maintain cellular homeostasis .

How are phospho-EIF2S1 (S51) antibodies contributing to understanding disease mechanisms?

Phospho-EIF2S1 (S51) antibodies have become valuable tools for investigating disease mechanisms across multiple pathological conditions:

Recombinant monoclonal antibodies with consistent performance characteristics are particularly valuable for translational research, as they enable reliable comparisons between different patient cohorts and experimental models . By correlating phospho-EIF2S1 (S51) levels with disease progression and treatment responses, researchers can identify potential intervention points in the integrated stress response pathway.

What novel assay technologies are being developed for phospho-EIF2S1 (S51) research?

The field of phospho-EIF2S1 (S51) research is witnessing the development of innovative assay technologies that enhance detection sensitivity, throughput, and multiplexing capabilities:

• Single-cell analysis approaches:

  • Phospho-flow cytometry for heterogeneity assessment

  • Single-cell Western blot techniques

  • Mass cytometry (CyTOF) with phospho-EIF2S1 antibodies

  • Imaging mass cytometry for spatial resolution

• Live-cell monitoring systems:

  • FRET-based biosensors for real-time EIF2S1 phosphorylation dynamics

  • Split luciferase complementation assays

  • Optogenetic control of EIF2S1 kinases

  • Integration with microfluidic platforms for temporal stress induction

• High-throughput screening applications:

  • Automated immunofluorescence microscopy

  • In-cell Western techniques

  • AlphaLISA or HTRF-based detection methods

  • Multiplex bead-based assays for simultaneous pathway analysis

• Spatial biology approaches:

  • Multiplexed immunofluorescence in tissues

  • Digital spatial profiling with phospho-specific antibodies

  • Correlation with single-cell transcriptomics

  • Whole-organism imaging in model systems

These technological advances facilitate more comprehensive analysis of EIF2S1 phosphorylation in complex biological systems, bridging the gap between molecular mechanisms and physiological outcomes .