EIF2S1 Antibody

What is EIF2S1 and what is its role in cellular processes?

EIF2S1 (eukaryotic translation initiation factor 2 subunit 1), also known as eIF2α, is a critical component of the translation initiation factor EIF2 complex that catalyzes the first regulated step of protein synthesis initiation. EIF2S1 plays a central role in cellular stress responses through its phosphorylation at Serine 51, which stabilizes the eIF-2/GDP/eIF-2B complex and prevents the GDP/GTP exchange reaction. This impairs the recycling of eIF-2 between successive rounds of initiation, leading to global inhibition of translation while selectively enhancing translation of specific stress-responsive mRNAs. EIF2S1 serves as a substrate for at least four stress-responsive kinases: EIF2AK1/HRI, EIF2AK2/PKR, EIF2AK3/PERK, and EIF2AK4/GCN2, which phosphorylate it in response to various cellular stresses including amino acid starvation and UV irradiation .

What types of EIF2S1 antibodies are available for research?

Researchers have access to several types of EIF2S1 antibodies, including:

• Total EIF2S1 antibodies: Detect EIF2S1 regardless of phosphorylation status

• Phospho-specific antibodies: Detect EIF2S1 only when phosphorylated at specific sites (e.g., Ser51)

• Species-specific variants: Available for human, mouse, rat, and other mammalian systems

• Clonality options:

  • Monoclonal antibodies (e.g., mouse monoclonal antibody [EIF2a] ab5369)

  • Polyclonal antibodies (e.g., rabbit polyclonal antibody 11170-1-AP)

  • Recombinant monoclonal antibodies (e.g., rabbit recombinant monoclonal IgG PSH04-29)

These antibodies come in various formats suited for different experimental techniques, including unconjugated forms for standard applications and some conjugated versions for specialized detection methods .

What are the common applications for EIF2S1 antibodies?

EIF2S1 antibodies have been validated for multiple experimental applications with specific working dilutions, as shown in the table below:

Each application requires specific sample preparation protocols and antigen retrieval methods. For instance, IHC applications frequently use heat-mediated antigen retrieval in citrate buffer (pH 6.0) or TE buffer (pH 9.0) before antibody incubation .

How should I optimize antibody dilution for Western blot detection of EIF2S1?

Optimizing antibody dilution for EIF2S1 detection requires a systematic approach:

• Initial dilution range test: Start with a broad range based on manufacturer recommendations (e.g., 1:1000, 1:5000, and 1:10000)

• Protein loading: Load 10-20 μg of total protein per lane for cell lysates (as used in validated examples)

• Positive controls: Include known positive samples such as MCF-7, HepG2, or Jurkat cell lysates

• Blocking conditions: Use 5% non-fat milk or BSA in TBST (samples may respond differently)

• Incubation time: Test both overnight incubation at 4°C and 1-2 hour incubation at room temperature

• Fine-tuning: Based on initial results, narrow the dilution range to optimize signal-to-noise ratio

The expected molecular weight for EIF2S1 is 36 kDa. Higher dilutions (1:10000-1:50000) often work well for total EIF2S1 detection, while phospho-specific antibodies may require lower dilutions (1:1000-1:5000). Always run appropriate negative controls and consider using EIF2S1 knockout/knockdown samples if available to confirm specificity .

What are the critical steps for successful immunofluorescence staining with EIF2S1 antibodies?

Successful immunofluorescence staining with EIF2S1 antibodies involves several critical steps:

• Fixation: Use 4% formaldehyde/paraformaldehyde for 10-15 minutes at room temperature

• Permeabilization: Apply 0.1-0.3% Triton X-100 for 5-10 minutes to allow antibody access to cytoplasmic EIF2S1

• Blocking: Block with 1-5% BSA combined with 10% normal serum (matching secondary antibody host) for 1 hour

• Primary antibody: Apply EIF2S1 antibody at 1:50-1:500 dilution, incubate overnight at 4°C or 1-2 hours at room temperature

• Secondary antibody: Use fluorophore-conjugated secondary (e.g., Alexa Fluor 488 or 594) at 1:500-1:1000 dilution

• Counterstaining: Include DAPI (1-5 μg/ml) for nuclear visualization

• Controls: Include a no-primary antibody control and, if possible, a siRNA knockdown control

For optimal results, include additional markers that can provide context for EIF2S1 localization. For example, in stress response studies, co-staining with stress granule markers can help visualize EIF2S1 recruitment to these structures. Expected EIF2S1 staining pattern is primarily cytoplasmic, with potential enrichment in stress granules under stress conditions .

How can I detect phosphorylated EIF2S1 in my experimental system?

Detecting phosphorylated EIF2S1 (p-EIF2S1) requires specific considerations:

• Antibody selection: Use phospho-specific antibodies that recognize EIF2S1 only when phosphorylated at Serine 51

• Positive control: Include samples treated with known inducers of EIF2S1 phosphorylation:

  • Thapsigargin (1-2 μM, 1-4 hours) for ER stress induction

  • Sodium arsenite (0.5 mM, 30-60 minutes) for oxidative stress

  • Interferon-alpha for PKR activation (as seen in K562 cells)

• Phosphatase inhibitors: Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in lysis buffers

• Sample handling: Process samples quickly and keep them cold to prevent dephosphorylation

• Loading controls: Run parallel blots for total EIF2S1 to calculate the p-EIF2S1/total EIF2S1 ratio

• Visualization: For immunofluorescence, dual staining with total EIF2S1 and p-EIF2S1 antibodies from different species can show co-localization

The p-EIF2S1 signal intensity typically increases in a dose-dependent manner with stress inducers. For Western blot, a time-course experiment (0, 15, 30, 60, 120 minutes) after stress induction can help identify the optimal time point for p-EIF2S1 detection .

Why might I be getting inconsistent results with my EIF2S1 antibody?

Inconsistent results with EIF2S1 antibodies can stem from several factors:

• Antibody degradation: Repeated freeze-thaw cycles can reduce antibody efficacy. Aliquot antibodies upon receipt and store at -20°C.

• Lot-to-lot variability: Different production lots may show variation. Document lot numbers used for critical experiments.

• Buffer incompatibility: Some sample buffers may not be compatible with the antibody. Test alternative buffers.

• Cell/tissue-specific expression levels: EIF2S1 expression varies across tissues and cell types. MCF-7, HepG2, and brain tissue typically show strong expression.

• Post-translational modifications: EIF2S1 undergoes various modifications beyond phosphorylation. Consider using antibodies specific to your modification of interest.

• Fixation artifacts: Overfixation can mask epitopes. Optimize fixation time for immunostaining applications.

• Antigen retrieval methods: For IHC, compare citrate buffer (pH 6.0) versus TE buffer (pH 9.0) for optimal results.

To troubleshoot, systematically test different conditions while changing only one variable at a time. Include positive controls (such as MCF-7 or HepG2 cell lysates) in each experiment. For phospho-specific antibodies, always include a stress-induced positive control sample .

How can I distinguish between specific and non-specific bands in Western blot?

Distinguishing between specific and non-specific bands when working with EIF2S1 antibodies requires several validation approaches:

• Molecular weight verification: The expected molecular weight for EIF2S1 is approximately 36 kDa. Bands significantly deviating from this size are likely non-specific.

• Positive controls: Compare your sample with validated positive controls like MCF-7, HepG2, or Jurkat cell lysates.

• Blocking peptide competition: Pre-incubating the antibody with its immunizing peptide should eliminate specific bands.

• Multiple antibodies: Test different antibodies against EIF2S1 (targeting different epitopes) to confirm consistent banding patterns.

• Genetic validation: If possible, use EIF2S1 knockdown/knockout samples as negative controls.

• Treatment response: Specific bands should respond appropriately to treatments (e.g., p-EIF2S1 increasing after stress induction).

• Cross-species reactivity: EIF2S1 is highly conserved; confirming expected bands across species can support specificity.

Some published validation data shows that in IFN-alpha treated K562 whole cell lysates, pre-incubation with the antigen-specific peptide effectively blocks detection of the specific EIF2S1 band, confirming antibody specificity .

What sample preparation methods optimize phospho-EIF2S1 detection?

Optimizing sample preparation for phospho-EIF2S1 detection is crucial for accurate results:

• Rapid sample processing: Minimize the time between cell/tissue collection and protein extraction to prevent dephosphorylation.

• Cold processing: Keep samples and buffers cold (on ice) throughout the preparation process.

• Phosphatase inhibitor cocktail: Include both serine/threonine and tyrosine phosphatase inhibitors:

  • 10 mM sodium fluoride

  • 1 mM sodium orthovanadate (activated)

  • 10 mM β-glycerophosphate

  • 1 mM PMSF

  • Commercial phosphatase inhibitor cocktails

• Lysis buffer selection: Use RIPA or NP-40 based buffers with phosphatase inhibitors.

• Protein quantification: Accurately quantify protein concentration to ensure equal loading.

• Sample storage: Store lysates at -80°C with phosphatase inhibitors; avoid repeated freeze-thaw cycles.

• Positive control treatments: Include samples treated with:

  • Thapsigargin (1-2 μM, 1-4 hours)

  • Tunicamycin (5 μg/ml, 4-8 hours)

  • Arsenite (0.5 mM, 30-60 minutes)

For time-course experiments analyzing stress responses, prepare a larger batch of cells and treat aliquots for different time periods rather than setting up separate experiments to reduce technical variability .

How can I use EIF2S1 antibodies to study the integrated stress response in different cell types?

EIF2S1 phosphorylation serves as a convergence point for various stress pathways in the integrated stress response (ISR). To study this system across cell types:

• Select appropriate stress inducers for specific EIF2S1 kinases:

  • PERK (EIF2AK3): Thapsigargin (1-2 μM) or tunicamycin (5 μg/ml) for ER stress

  • PKR (EIF2AK2): Poly(I:C) transfection or viral infection

  • HRI (EIF2AK1): Sodium arsenite (0.5 mM) for oxidative stress

  • GCN2 (EIF2AK4): Amino acid starvation media or halofuginone

• Multiplex detection approach:

  • Western blot: Probe for p-EIF2S1, total EIF2S1, and downstream factors (ATF4, CHOP)

  • Immunofluorescence: Co-stain for p-EIF2S1 and stress-specific markers

  • RT-qPCR: Measure ISR target gene expression (ATF4, CHOP, GADD34)

• Cell type-specific considerations:

  • Neuronal cells: More sensitive to PERK activation

  • Hepatocytes: Strong GCN2 responses

  • Immune cells: Prominent PKR signaling

  • Erythroid precursors: HRI plays a major role

Analyzing the kinetics of EIF2S1 phosphorylation and dephosphorylation across cell types can reveal tissue-specific stress response mechanisms. This approach has been successfully implemented in comparing responses between wild-type and phosphorylation-deficient cells, revealing differences in autophagy and UPR gene expression patterns during ER stress .

What experimental approaches can reveal the relationship between EIF2S1 phosphorylation and autophagy?

Recent research has established connections between EIF2S1 phosphorylation and autophagy regulation. To investigate this relationship:

• Genetic tools:

  • Use EIF2S1 S51A phosphorylation-deficient cells (knock-in mutants)

  • Compare with wild-type cells under various stress conditions

  • Employ kinase-specific knockout models (PERK, GCN2, etc.)

• Autophagy monitoring:

  • LC3-II Western blot with/without lysosomal inhibitors

  • GFP-LC3 puncta formation assay

  • Autophagic flux assays with tandem mRFP-GFP-LC3

  • Transmission electron microscopy for autophagosome visualization

• Transcriptional analysis:

  • RT-qPCR for autophagy genes in WT vs. phospho-deficient cells

  • ChIP assays to detect transcription factor binding to autophagy gene promoters

  • RNA-seq to identify global transcriptional changes

• Rescue experiments:

  • Express constitutively active forms of downstream effectors (ATF4, ATF6, XBP1s, TFEB)

  • Assess their ability to restore autophagy in phosphorylation-deficient cells

Research has shown that EIF2S1 phosphorylation-deficient cells (S51A mutants) exhibit dysregulated expression of autophagy genes during ER stress. Overexpression of activated ATF6 or TFEB can more efficiently rescue autophagic defects in these cells, though XBP1s and ATF4 also show some ability to restore autophagy, suggesting complex interplay between UPR pathways and autophagy regulation .

How can I design experiments to study EIF2S1's role in stress granule formation?

EIF2S1 phosphorylation plays a critical role in stress granule (SG) assembly. Design experiments to explore this connection:

• Stress conditions and visualization:

  • Apply arsenite (0.5 mM, 30-60 min), thapsigargin (1 μM, 1 hour), or heat shock (42°C, 1 hour)

  • Co-immunostain for p-EIF2S1 and SG markers (G3BP1, TIA-1, PABP)

  • Use live-cell imaging with fluorescently tagged SG components

• Genetic manipulations:

  • Compare SG formation in wild-type vs. EIF2S1-S51A mutant cells

  • Use siRNA/shRNA against EIF2S1 kinases to determine which stress pathways affect SG dynamics

  • Express phosphomimetic EIF2S1-S51D to test if phosphorylation is sufficient for SG induction

• Quantitative analysis:

  • Measure SG size, number, and composition across conditions

  • Assess kinetics of assembly and disassembly

  • Correlate p-EIF2S1 levels with SG parameters

• Therapeutic modulation:

  • Test ISRIB (Integrated Stress Response Inhibitor) effects on SG formation

  • Apply salubrinal to inhibit eIF2α dephosphorylation and examine SG persistence

  • Investigate how modulating p-EIF2S1 levels affects SG-associated pathologies

• Interaction studies:

  • Perform co-IP experiments to identify stress-dependent interactions

  • Use proximity ligation assays to detect in situ associations

  • Apply FRAP (Fluorescence Recovery After Photobleaching) to assess protein dynamics within SGs

These approaches can help elucidate how EIF2S1 phosphorylation regulates the composition and dynamics of stress granules in different cellular contexts, potentially revealing therapeutic targets for stress-related disorders .

How should I normalize and quantify phospho-EIF2S1 Western blot data?

Proper normalization and quantification of phospho-EIF2S1 (p-EIF2S1) Western blot data ensures accurate interpretation of results:

When analyzing stress responses, remember that p-EIF2S1 levels typically show dynamic changes over time. For example, ER stress may cause rapid phosphorylation (within 15-30 minutes) followed by adaptive feedback mechanisms that reduce phosphorylation levels despite continued stress exposure .

What controls are essential when analyzing EIF2S1 phosphorylation in disease models?

When studying EIF2S1 phosphorylation in disease models, include these essential controls:

• Positive and negative controls:

  • Positive control: Samples treated with known inducers of EIF2S1 phosphorylation (thapsigargin, arsenite)

  • Negative control: Phosphatase-treated lysates to establish baseline signal

  • Genetic controls: When possible, include EIF2S1-S51A mutant cells/tissues

• Technical validation controls:

  • Antibody specificity: Validate with blocking peptides or phosphatase treatment

  • Cross-reactivity: Test antibodies against related phosphoproteins

  • Loading consistency: Verify with total protein stains and housekeeping proteins

• Biological context controls:

  • Time-matched controls: Account for circadian or cell-cycle variations

  • Vehicle controls: For drug treatments, include appropriate vehicle-only conditions

  • Environmental controls: Match temperature, humidity, feeding conditions for animal models

• Disease-specific considerations:

  • Disease progression: Sample at multiple stages of pathology

  • Genetic background matching: Ensure control and disease models share genetic background

  • Age and sex matching: Control for age and sex-specific differences in stress responses

  • Therapeutic intervention: Include treated and untreated disease models

• Validation across methodologies:

  • Confirm key findings using multiple detection methods (WB, IHC, IF)

  • Consider mass spectrometry validation for phosphorylation site specificity

  • Correlate protein changes with functional readouts (translation attenuation, ATF4 induction)

These controls are particularly important when studying diseases associated with EIF2S1 dysregulation, including neurodegenerative disorders, diabetes, and various cancers, where altered stress responses may contribute to pathology .

How can I integrate EIF2S1 phosphorylation data with other UPR pathway markers?

Integrating EIF2S1 phosphorylation data with other UPR markers provides a comprehensive view of cellular stress responses:

• Multi-pathway analysis approach:

  • PERK pathway: p-EIF2S1, total EIF2S1, ATF4, CHOP, GADD34

  • IRE1 pathway: XBP1 splicing, p-IRE1α, ERDJ4, P58IPK

  • ATF6 pathway: Cleaved ATF6, GRP78/BiP, ERSE-regulated genes

  • Integrated readouts: Cell viability, apoptosis markers, protein synthesis rates

• Combined methodologies:

  • Protein analysis: Multiplex Western blots or flow cytometry for UPR proteins

  • Transcriptional profiling: RT-qPCR for UPR target genes or RNA-seq for global changes

  • Reporter assays: CHOP-GFP, XBP1s-GFP, or ATF6-responsive elements

  • Functional assays: Puromycin incorporation for translation, annexin V for apoptosis

• Data integration strategies:

  • Correlation analysis: Determine relationships between different UPR markers

  • Principal component analysis: Identify major patterns in UPR activation

  • Cluster analysis: Group samples based on UPR activation profiles

  • Pathway scoring: Develop composite scores for each UPR branch

• Temporal considerations:

  • Include multiple time points (early: 0.5-2h, intermediate: 4-8h, late: 12-24h)

  • Track the sequential activation of different UPR branches

  • Monitor adaptive versus terminal UPR responses

• Visualization approaches:

  • Heat maps showing all UPR markers across conditions

  • Radar plots displaying the relative activation of each UPR branch

  • Network diagrams illustrating interconnections between pathways

Research has shown that EIF2S1 phosphorylation connects UPR pathways to autophagy, with phosphorylation-deficient cells showing dysregulated expression of both UPR and autophagy genes during ER stress. Integrating data across these pathways can reveal how cells coordinate different adaptive responses to restore homeostasis .

What are the most influential papers describing EIF2S1 function in stress responses?

While I cannot provide a comprehensive literature review, several key publications have shaped our understanding of EIF2S1 function in stress responses. Researchers should consult these foundational papers:

• Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 1999.

  • Established PERK as an ER stress-responsive kinase that phosphorylates EIF2S1

• Wek RC, Jiang H-Y, Anthony TG. Coping with stress: eIF2 kinases and translational control. Biochemical Society Transactions. 2006.

  • Comprehensive review of the four eIF2α kinases and their roles in stress responses

• Harding HP, Novoa I, Zhang Y, et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Molecular Cell. 2000.

  • Demonstrated how EIF2S1 phosphorylation paradoxically increases translation of ATF4

• Scheuner D, Song B, McEwen E, et al. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Molecular Cell. 2001.

  • Used EIF2S1 S51A knock-in mice to demonstrate physiological importance of phosphorylation

• Taniuchi S, Miyake M, Tsugawa K, et al. Integrated stress response of vertebrates is regulated by four eIF2α kinases. Scientific Reports. 2016.

  • Comprehensive analysis of the four EIF2S1 kinases and their specific and overlapping functions

More recent research has established connections between EIF2S1 phosphorylation and autophagy, demonstrating that this modification is important for both autophagy and UPR pathways in restoring ER homeostasis .

Where can I find validated protocols for using EIF2S1 antibodies in various applications?

Researchers can access validated protocols for EIF2S1 antibodies from several reliable sources:

• Commercial antibody providers:

  • Detailed datasheets with application-specific protocols from vendors like Proteintech, Abcam, and Cell Signaling Technology

  • Online resources including troubleshooting guides and video tutorials

• Method-specific repositories:

  • Protocols.io: Peer-reviewed protocols for various applications

  • Bio-protocol: Curated, step-by-step protocols often linked to published papers

  • Journal of Visualized Experiments (JoVE): Video protocols demonstrating techniques

• Published literature:

  • Materials and methods sections of papers using EIF2S1 antibodies

  • Supplementary protocols from key papers in the field

• Research resource identification:

  • Use RRID numbers (e.g., AB_2096489 for Proteintech's 11170-1-AP antibody) to find publications using specific antibodies

• Collaborative platforms:

  • Research Gate and other scientific forums where researchers share optimized protocols

  • Lab websites of prominent stress response researchers

When adapting protocols, pay special attention to sample preparation methods, antigen retrieval techniques for fixed samples, and recommended dilutions for specific applications. For example, Proteintech's antibody (11170-1-AP) has been validated for WB at 1:5000-1:50000 dilution, while IHC applications typically require 1:50-1:500 dilution with specific antigen retrieval methods .

How are EIF2S1 antibodies being used to study stress granule dynamics in neurodegenerative diseases?

EIF2S1 phosphorylation and stress granule formation have emerged as important factors in neurodegenerative diseases. Researchers are using EIF2S1 antibodies in several innovative approaches:

• Post-mortem tissue analysis:

  • Comparing p-EIF2S1 levels in brain regions from patients with Alzheimer's, Parkinson's, and ALS versus controls

  • Co-localization studies of p-EIF2S1 with pathological protein aggregates (tau, α-synuclein, TDP-43)

  • Correlating p-EIF2S1 levels with disease progression markers

• Disease models:

  • Tracking p-EIF2S1 and stress granule dynamics in patient-derived iPSCs differentiated into neurons

  • Studying animal models expressing disease-associated mutations (e.g., SOD1, TDP-43, tau)

  • Real-time imaging of stress granule formation in neuronal cultures under proteotoxic stress

• Therapeutic interventions:

  • Testing compounds that modulate EIF2S1 phosphorylation (ISRIB, salubrinal) in disease models

  • Assessing whether preventing pathological stress granule formation can mitigate neurodegeneration

  • Using genetic approaches to manipulate EIF2S1 phosphorylation in specific neuronal populations

• Mechanistic investigations:

  • Examining how disease-associated mutations affect EIF2S1 phosphorylation kinetics

  • Studying the interplay between stress granules and other cellular compartments (autophagosomes, mitochondria)

  • Investigating cell-type specific responses in neurons versus glia

These approaches may reveal how chronic stress pathway activation contributes to neurodegeneration and identify potential therapeutic targets for intervention. Researchers should consider using multiple antibodies to validate findings and include appropriate controls when studying post-mortem tissue .

What role does EIF2S1 phosphorylation play in cancer development and therapy resistance?

EIF2S1 phosphorylation has complex and sometimes contradictory roles in cancer biology:

• Dual functions in cancer progression:

  • Tumor suppressive: Sustained EIF2S1 phosphorylation can trigger apoptosis through CHOP induction

  • Tumor promoting: Transient/moderate phosphorylation can promote survival under stress conditions

  • Metabolic adaptation: Helps cancer cells survive nutrient limitation and hypoxia

• Experimental approaches to study EIF2S1 in cancer:

  • Compare p-EIF2S1 levels across cancer types and stages using tissue microarrays

  • Correlate p-EIF2S1 with patient outcomes and treatment responses

  • Manipulate EIF2S1 phosphorylation in cancer cells to assess effects on proliferation, migration, and drug sensitivity

• Therapy resistance mechanisms:

  • Cancer cells often adapt to therapy-induced stress through EIF2S1 phosphorylation

  • This promotes selective translation of survival factors and stress-adaptive proteins

  • Combined inhibition of stress adaptation pathways with conventional therapies may overcome resistance

• Experimental design considerations:

  • Use multiple cancer cell lines representing different cancer types and genetic backgrounds

  • Test effects of EIF2S1 phosphorylation modulators on response to standard chemotherapeutics

  • Develop xenograft models to assess in vivo relevance of findings

Experimental data from breast cancer cell lines (MCF-7 and MDA-MB-231) shows that treatment with BA (butyric acid) induces ER stress-associated signals in a dose-dependent manner, including increased p-EIF2S1/EIF2S1 ratio, along with GRP78, p-PERK/PERK, CHOP, and caspase-12 activation. This suggests that modulating the EIF2S1 phosphorylation pathway might sensitize cancer cells to certain treatments .

How can multiplexed detection methods be used to analyze EIF2S1 in the context of integrated stress responses?

Advanced multiplexed detection approaches offer powerful tools for analyzing EIF2S1 in the broader context of integrated stress responses:

• Multiplexed immunofluorescence techniques:

  • Cyclic immunofluorescence (CycIF): Sequential staining with different antibodies and fluorophore inactivation

  • Multiplexed ion beam imaging (MIBI): Metal-tagged antibodies detected by mass spectrometry

  • CO-Detection by indEXing (CODEX): DNA-barcoded antibodies with sequential fluorophore exposure

  • These methods can simultaneously detect p-EIF2S1, total EIF2S1, and multiple stress pathway components

• Mass cytometry approaches:

  • CyTOF (Cytometry by Time-Of-Flight): Metal-labeled antibodies for single-cell multiparameter analysis

  • scMS-proteomics: Single-cell mass spectrometry to detect phosphorylation events

  • These techniques allow correlation of p-EIF2S1 with dozens of other cellular parameters

• Spatial transcriptomics integration:

  • Combine p-EIF2S1 immunostaining with spatial transcriptomics

  • Map stress response gene expression patterns relative to p-EIF2S1 distribution

  • Identify microenvironmental factors influencing EIF2S1 phosphorylation

• Live-cell multiparameter imaging:

  • Fluorescent biosensors for translation rates combined with p-EIF2S1 reporters

  • Simultaneous tracking of multiple stress response pathways

  • Correlative light and electron microscopy to connect molecular events to ultrastructural changes

• Experimental design considerations:

  • Include appropriate single-stained controls for compensation/unmixing

  • Use isotype controls and phosphatase-treated samples as controls

  • Apply computational methods (clustering, dimensionality reduction) to identify stress response patterns