ERP27 Antibody

What is ERP27 and what is its primary function in cellular physiology?

ERP27 (Endoplasmic Reticulum Protein 27) is a protein localized to the endoplasmic reticulum that specifically binds to unfolded proteins. Despite lacking the typical thioredoxin-like active sites found in many ER-resident proteins, ERP27 plays a significant role in protein maturation by assisting in the correct assembly and folding of nascent proteins. It functions primarily by recruiting protein disulfide isomerase PDIA3 to unfolded substrates and may play a crucial role in the unfolded protein stress response . This protein contains a hydrophobic pocket in its C-terminal domain that facilitates substrate binding . ERP27 is also known by several alternative names including C12orf46, UNQ781/PRO1575, ER protein 27, ERp27, and inactive protein disulfide-isomerase 27 .

Which ERP27 antibody formats are available for research applications?

Several formats of ERP27 antibodies are available for research applications, primarily categorized as polyclonal and monoclonal antibodies with varying species reactivity:

These antibody formats offer researchers flexibility in experimental design based on their specific application requirements .

How does ERP27 differ structurally and functionally from other PDI family members?

Although ERP27 is sometimes classified within the protein disulfide isomerase (PDI) family (as PDIA8), it differs significantly from typical PDI members. Unlike canonical PDI family proteins, ERP27 lacks the characteristic thioredoxin-like active sites necessary for catalytic disulfide bond formation or isomerization . Instead, ERP27 functions primarily as a non-catalytic binding partner that recognizes unfolded protein substrates through its hydrophobic pocket and subsequently recruits enzymatically active PDI family members like PDIA3 . This structural difference positions ERP27 as a specialized adapter protein within the ER quality control system rather than as a direct catalyst of protein folding.

What are the optimal conditions for using ERP27 antibodies in Western blotting experiments?

For Western blotting applications using ERP27 antibodies, researchers should follow these optimized protocol parameters:

• Sample preparation: Human pancreas tissue lysate shows strong endogenous ERP27 expression and serves as an excellent positive control .

• Antibody dilution: The recommended working dilution for most anti-ERP27 antibodies in Western blotting is 1:1000 . This concentration provides optimal signal-to-noise ratio for detection of the predicted 30 kDa ERP27 band.

• Secondary antibody selection: Anti-rabbit IgG HRP-conjugated secondary antibodies at a dilution of 1:500 to 1:1000 work effectively with rabbit-derived primary ERP27 antibodies .

• Protein loading: 10-20 μg of total protein per lane typically provides sufficient target protein for detection with minimal background .

• Controls: Include mouse and rat pancreas lysates as additional controls when working with antibodies that cross-react with these species .

These conditions have been experimentally validated and shown to produce clear detection of the predicted 30 kDa ERP27 protein band .

How should researchers optimize immunocytochemistry protocols for ERP27 detection?

For optimal immunocytochemical detection of ERP27, researchers should consider these methodological parameters:

• Fixation: 4% paraformaldehyde fixation preserves both antigenicity and cellular architecture for ERP27 detection .

• Antibody dilution: A working dilution of 1:250 for primary ERP27 antibodies provides excellent signal with minimal background in ICC applications .

• Secondary antibody selection: Fluorophore-conjugated anti-rabbit antibodies (e.g., Alexa Fluor® 555) at 1:200 dilution provide strong visualization of the target protein .

• Cell type considerations: BxPC-3 cells (human pancreatic cancer cells) demonstrate good endogenous expression of ERP27 and are recommended as positive controls for ICC protocol optimization .

• Counterstaining: DAPI nuclear counterstaining allows clear visualization of subcellular localization of ERP27, which typically displays an ER-characteristic reticular pattern consistent with its biological function .

This protocol has been validated to produce specific labeling of ERP27 with typical ER morphology pattern and minimal non-specific background staining .

What are the key considerations for flow cytometry applications using ERP27 antibodies?

When implementing flow cytometry protocols for intracellular detection of ERP27, researchers should consider these critical parameters:

• Cell fixation and permeabilization: 2% paraformaldehyde fixation followed by appropriate permeabilization is essential for accessing intracellular ERP27 .

• Antibody concentration: A higher antibody concentration (1:50 dilution) is typically required for flow cytometry compared to other applications .

• Cell selection: HeLa cells show detectable ERP27 expression and serve as suitable positive controls for protocol optimization .

• Controls: Include appropriate isotype controls (e.g., rabbit monoclonal IgG at matching concentration) to establish proper gating and confirm specificity .

• Secondary antibody: FITC-conjugated anti-rabbit IgG secondary antibodies at 1:150 dilution provide good signal amplification while maintaining specificity .

This approach enables reliable quantification of intracellular ERP27 expression across cell populations with minimal non-specific binding .

How can researchers differentiate between specific and non-specific binding when using ERP27 antibodies?

Differentiating between specific and non-specific binding when using ERP27 antibodies requires a multi-faceted validation approach:

• Molecular weight verification: Confirm detection of the predicted 30 kDa band in Western blotting applications, as aberrant band patterns may indicate non-specific binding .

• Tissue expression pattern analysis: Compare observed staining patterns with known ERP27 expression profiles. ERP27 shows highest expression in secretory tissues like pancreas, making these ideal positive controls. Unexpected staining in tissues with no documented ERP27 expression suggests non-specificity .

• Subcellular localization assessment: Authentic ERP27 staining should display endoplasmic reticulum-specific patterns. Diffuse cytoplasmic, nuclear, or membranous staining patterns without ER characteristics likely represent non-specific binding .

• Competitive binding assays: Pre-incubation of antibodies with purified ERP27 protein should abolish specific signals while leaving non-specific binding unaffected.

• Correlation across detection methods: Consistent detection patterns across multiple techniques (WB, IHC, ICC) increases confidence in specificity. Discrepancies between methods warrant further investigation of potential non-specific interactions.

Implementing these validation steps systematically enhances confidence in experimental results and facilitates accurate interpretation of ERP27 localization and function studies .

What approaches can researchers use to study ERP27 interactions with PDIA3 and other binding partners?

To effectively investigate ERP27's interactions with PDIA3 and other potential binding partners, researchers can employ these methodological approaches:

• Co-immunoprecipitation (Co-IP): Using anti-ERP27 antibodies to pull down protein complexes followed by Western blotting for PDIA3 or other suspected partners. This approach requires carefully optimized antibody concentrations and washing conditions to preserve weak or transient interactions.

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity and specificity, allowing detection of ERP27-PDIA3 complexes within their native cellular compartments.

• Fluorescence resonance energy transfer (FRET): By tagging ERP27 and potential binding partners with appropriate fluorophore pairs, researchers can detect direct molecular interactions through energy transfer measurements.

• Bimolecular fluorescence complementation (BiFC): This approach involves fusing complementary fragments of a fluorescent protein to ERP27 and its putative binding partners, enabling visualization of interactions through reconstitution of fluorescence.

• Mass spectrometry following cross-linking: Chemical cross-linking of protein complexes followed by mass spectrometric analysis can identify novel binding partners and characterize interaction interfaces, particularly valuable for identifying components of larger multiprotein complexes involving ERP27.

Each of these approaches offers distinct advantages for studying different aspects of ERP27's protein interaction network and should be selected based on the specific research question being addressed .

How does ERP27 expression correlate with unfolded protein response activation in different experimental models?

The correlation between ERP27 expression and unfolded protein response (UPR) activation appears to be context-dependent across experimental models:

• Stress induction dynamics: ERP27 expression patterns in response to classic UPR inducers (tunicamycin, thapsigargin, DTT) show tissue-specific and temporal variations. Unlike canonical UPR markers like BiP/GRP78 that show rapid induction, ERP27 upregulation may occur with different kinetics depending on the cell type and stressor.

• Pathological conditions: In disease models characterized by chronic ER stress (neurodegenerative disorders, diabetes models), ERP27 expression changes may correlate with disease progression and severity, potentially serving as a biomarker for sustained rather than acute UPR activation.

• Cancer models: Several pancreatic cancer cell lines show altered ERP27 expression patterns compared to normal pancreatic tissue, suggesting a potential role in adaptive responses to the heightened protein folding demands in rapidly proliferating cells .

• Correlation with UPR branches: ERP27 expression may correlate differentially with specific UPR branches (PERK, IRE1, ATF6), providing insights into its functional role within the broader stress response network.

• Recovery phase monitoring: Tracking ERP27 levels during the recovery phase following ER stress resolution may reveal distinct patterns compared to classic UPR mediators, potentially indicating specialized roles in the restoration of normal ER function.

These diverse response patterns highlight the complexity of ERP27's role within the broader ER quality control system and suggest that its regulation may involve both UPR-dependent and independent mechanisms .

What are common challenges in immunohistochemistry using ERP27 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when performing immunohistochemistry with ERP27 antibodies. These issues and their solutions include:

• Antigen retrieval optimization: For paraffin-embedded tissues, heat-mediated antigen retrieval with EDTA buffer (pH 9.0) significantly improves ERP27 detection compared to citrate buffer methods . Insufficient retrieval often manifests as weak or absent staining, particularly in densely fixed tissues.

• Background reduction strategies: Non-specific background staining can be minimized through:

  • Extended blocking (1-2 hours) with 5% normal serum from the same species as the secondary antibody

  • Addition of 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Careful antibody titration to identify the optimal concentration that maximizes specific signal while minimizing background

• Tissue-specific considerations: Pancreatic tissue often shows strong endogenous ERP27 expression but may present challenges due to high endogenous peroxidase activity. Extended peroxidase blocking (15-20 minutes with 3% H₂O₂) is recommended for these tissues .

• Signal amplification methods: For tissues with low ERP27 expression, polymer-based detection systems provide superior sensitivity compared to traditional ABC methods while maintaining specificity .

• Counterstain optimization: Hematoxylin counterstaining time should be carefully controlled (30-60 seconds) to ensure nuclear visualization without obscuring specific cytoplasmic ERP27 staining .

Implementing these optimized approaches can significantly improve both the sensitivity and specificity of ERP27 detection in diverse tissue types .

How can researchers validate antibody specificity for ERP27 in their experimental systems?

A comprehensive validation strategy for ERP27 antibodies should include multiple complementary approaches:

• Multi-technique confirmation: Validate antibody performance across multiple techniques (WB, IHC, ICC) to ensure consistent detection patterns. ERP27 should consistently appear as a 30 kDa band in Western blots and display ER-specific staining patterns in microscopy applications .

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals while leaving non-specific binding unaffected, providing a powerful control for specificity.

• Genetic approaches: Where possible, validation in systems with genetic manipulation of ERP27 (knockout, knockdown, or overexpression) provides the most definitive confirmation of specificity. Signal intensity should correlate directly with ERP27 expression levels.

• Cross-species reactivity assessment: For antibodies claiming cross-reactivity with multiple species, parallel testing in human, mouse, and rat samples (particularly pancreatic tissue) allows confirmation of appropriate species recognition based on evolutionary conservation of epitopes .

• Epitope mapping: Understanding the specific epitope recognized by the antibody enables more informed experimental design and interpretation, particularly when studying protein domains with specific functions or interactions.

This multi-parameter validation approach ensures robust experimental outcomes and facilitates accurate interpretation of results across diverse research applications .

How should researchers interpret discrepancies in ERP27 detection between different antibodies or techniques?

When researchers encounter discrepancies in ERP27 detection patterns across different antibodies or techniques, systematic analysis should guide interpretation:

• Epitope accessibility considerations: Different antibodies may recognize distinct epitopes with varying accessibility in different experimental contexts. C-terminal epitopes might be masked in protein complexes, while N-terminal epitopes could be affected by signal peptide processing.

• Post-translational modification interference: Phosphorylation, glycosylation, or other modifications may selectively interfere with epitope recognition by certain antibodies, particularly in specific physiological or stress conditions.

• Isoform-specific detection: The human ERP27 gene may produce multiple protein isoforms through alternative splicing or post-translational processing. Different antibodies may preferentially detect specific isoforms, leading to apparently discrepant results.

• Method-specific artifacts: Each detection method has inherent limitations:

  • Western blotting excels at size discrimination but can miss conformational epitopes

  • Immunohistochemistry preserves spatial context but may suffer from fixation artifacts

  • Flow cytometry provides quantitative data but may be affected by permeabilization conditions

• Technical validation approach: When discrepancies arise, researchers should:

  • Compare results with literature reports of ERP27 expression patterns

  • Test multiple antibodies targeting different epitopes

  • Correlate protein detection with mRNA expression data

  • Consider alternative detection methods that don't rely on antibody recognition

This systematic troubleshooting approach allows researchers to distinguish between biologically meaningful variations and technical artifacts .

What are emerging research areas where ERP27 antibodies are providing new insights?

ERP27 antibody-based research is advancing understanding in several cutting-edge areas:

• ER stress dynamics in neurodegenerative diseases: Recent studies are utilizing ERP27 antibodies to investigate non-canonical ER stress responses in models of Alzheimer's, Parkinson's, and ALS, revealing potential disease-specific patterns distinct from classical UPR markers.

• Pancreatic disease progression: Given the high expression of ERP27 in pancreatic tissue, antibody-based studies are exploring its potential as a biomarker for early detection of pancreatic cancer and monitoring treatment responses .

• Secretory cell specialization: Comparative immunohistochemical analyses across diverse secretory cell types are revealing tissue-specific variations in ERP27 expression patterns, suggesting specialized roles in different secretory contexts.

• ER quality control network mapping: Advanced proteomics approaches using ERP27 antibodies for immunoprecipitation followed by mass spectrometry are uncovering previously unrecognized protein interaction networks within the ER quality control system.

• Therapeutic antibody development enhancement: Insights from ERP27's role in protein folding are informing strategies to improve the developability of therapeutic antibodies by enhancing folding efficiency and reducing aggregation propensity .

These emerging research directions highlight the continuing value of ERP27 antibodies as tools for uncovering fundamental mechanisms in protein homeostasis and their implications for health and disease .

How can researchers integrate ERP27 antibody data with other proteomic and genomic datasets?

Effective integration of ERP27 antibody-derived data with broader -omics datasets requires thoughtful analytical approaches:

• Correlation with transcriptomic profiles: ERP27 protein expression patterns detected by antibodies can be correlated with RNA-seq or microarray data to identify potential post-transcriptional regulatory mechanisms affecting protein abundance.

• Network analysis integration: ERP27 antibody-based interactome data can be incorporated into protein-protein interaction networks derived from high-throughput studies, positioning ERP27 within functional modules related to ER quality control.

• Multi-omics data visualization: Advanced visualization tools allow researchers to overlay ERP27 antibody-derived expression or localization data onto broader proteomic landscapes, revealing co-regulation patterns with functionally related proteins.

• Pathway enrichment analysis: Datasets incorporating ERP27 antibody results can be subjected to pathway enrichment analysis to identify biological processes that may be co-regulated with ERP27 under specific conditions.

• Machine learning approaches: Training machine learning algorithms on integrated datasets that include ERP27 antibody results alongside other -omics data can reveal non-obvious patterns and generate testable hypotheses about ERP27 function.

This integrative approach maximizes the value of ERP27 antibody data by contextualizing findings within the broader molecular landscape of the cell, potentially revealing emergent properties not apparent from isolated analyses .

What are the future prospects for ERP27 antibody development and application?

The evolution of ERP27 antibody technology is likely to advance along several promising trajectories:

• Epitope-specific antibodies: Development of antibodies targeting functional domains of ERP27 (particularly the substrate-binding hydrophobic pocket) could enable more nuanced studies of its interactions with client proteins and regulatory partners.

• Modification-specific antibodies: Generation of antibodies recognizing specific post-translational modifications of ERP27 would facilitate studies of its regulation under various physiological and stress conditions.

• Super-resolution microscopy applications: Optimization of ERP27 antibodies for emerging super-resolution techniques could reveal previously unrecognized spatial organization patterns within the ER folding machinery.

• In vivo imaging capabilities: Development of ERP27 antibody-based probes suitable for in vivo imaging could enable longitudinal studies of ER stress responses in disease models.

• Therapeutic potential: Greater understanding of ERP27's role in protein folding quality control may reveal opportunities for therapeutic intervention in diseases characterized by ER dysfunction, with antibody-derived molecules potentially serving as modulators of this pathway.

These advances will continue to expand the utility of ERP27 antibodies as both research tools and potential therapeutic development platforms .