PDIA3 Antibody

What is PDIA3 and why are PDIA3 antibodies important in research?

PDIA3 (Protein Disulfide Isomerase Family A Member 3), also known as ERp57, GRP58, or ER-60, is a pleiotropic member of the protein disulfide isomerase (PDI) family. It is predominantly located in the endoplasmic reticulum but has been detected in other cellular compartments, including the cell surface membrane .

PDIA3 antibodies are crucial research tools because:

• They enable detection of PDIA3 upregulation during stress conditions

• They allow investigation of PDIA3's translocation to cell surfaces during metabolic stress

• They help elucidate PDIA3's role in immunogenic cell death

• They are used to study PDIA3's involvement in various pathological conditions including liver inflammation, cancer progression, and muscle regeneration

Key functions of PDIA3 identified through antibody-based research:

• Chaperone activity for protein folding

• Cell surface signaling during stress responses

• Involvement in myoblast differentiation and fusion

• Potential autoantigen in inflammatory conditions

What applications are PDIA3 antibodies most commonly used for?

PDIA3 antibodies are versatile tools employed across multiple experimental applications:

When selecting applications, researchers should note that PDIA3 detection may require different optimization approaches depending on subcellular localization (intracellular vs. surface expression) .

What are the critical factors in validating PDIA3 antibodies for experimental use?

Validation is essential to ensure specificity and sensitivity of PDIA3 antibodies:

• Specificity confirmation methods:

  • siRNA knockdown validation (validate by observing decreased signal intensity after PDIA3 knockdown)

  • Western blot band identification at the expected molecular weight (~57-60 kDa)

  • Peptide mass fingerprinting of immunoprecipitated proteins

  • Cross-validation with multiple antibodies targeting different epitopes

• Application-specific validation:

  • For intracellular staining: Co-localization with ER markers

  • For surface staining: Validation with non-permeabilized live cells

  • For functional studies: Confirmation of blocking capacity via biological assay readouts

Remember that antibody validation should be performed for each specific application and experimental condition, as antibody performance can vary significantly across different contexts .

How should researchers properly prepare samples for PDIA3 antibody detection?

Sample preparation significantly impacts PDIA3 antibody detection success:

For Western blotting:

• Extract proteins using buffers containing protease inhibitors to prevent degradation

• Use reducing conditions (DTT or β-mercaptoethanol) for most applications

• Recommended loading: 10-20 μg total protein per lane

• Optimal protein transfer using 0.2 μm PVDF membranes for better retention of proteins

For immunofluorescence/immunohistochemistry:

• Fixation method matters: Paraformaldehyde (4%) preserves epitope accessibility

• For surface PDIA3 detection: Use live cell staining without permeabilization

• For intracellular PDIA3: Standard fixation and permeabilization protocols are effective

For studying secreted PDIA3:

• Collect conditioned media after 48h culture

• Remove cell debris by centrifugation (400 rcf, 5 min)

• Filter through 0.22 μm sterile filter

• Consider concentrating dilute samples

How can PDIA3 antibodies be used to investigate PDIA3's role in immunogenic cell death and autoimmunity?

PDIA3 has emerged as a potential bridge between immunogenic cell death (ICD) and autoimmunity, particularly in liver diseases. Research methodologies using PDIA3 antibodies include:

For studying PDIA3 as an autoantigen:

• ELISA-based detection of anti-PDIA3 autoantibodies in patient sera

• Isotype profiling of anti-PDIA3 antibodies (IgM vs. IgG3) to assess maturation of autoimmune responses

• Peptide epitope mapping using synthetic PDIA3 peptides to identify immunodominant regions

Experimental models for PDIA3 autoimmunity:

• High-fat high-fructose (HFHF) diet mouse models show PDIA3 surface expression on stressed hepatocytes

• Passive transfer experiments with purified anti-PDIA3 antibodies to assess pathogenicity

• T cell transfer studies with PDIA3-specific T cells to evaluate cellular immunity contributions

Research has shown that PDIA3-specific antibodies contribute to hepatotoxicity in metabolically stressed livers but not in healthy controls, suggesting a "second hit" requirement for pathogenicity . Detection of these autoantibodies in patients with autoimmune hepatitis, primary biliary cholangitis, and type 2 diabetes indicates potential clinical relevance .

What techniques can be used to study the cell surface translocation of PDIA3 during stress conditions?

PDIA3 translocation to the cell surface during stress is a critical research area with methodological considerations:

Methods for detecting surface PDIA3:

• Live cell immunofluorescence without permeabilization

• Cell surface biotinylation followed by streptavidin pulldown and PDIA3 immunoblotting

• Flow cytometry with non-permeabilized cells using anti-PDIA3 antibodies

• Co-staining with membrane markers (e.g., MG53) to confirm surface localization

Quantification approaches:

• Cell Surface Protein Isolation Kit followed by Western blotting to measure relative surface expression levels

• ELISA of culture supernatants to detect secreted PDIA3 during differentiation or stress

Research has demonstrated that PDIA3 surface expression increases during:

• Metabolic stress in hepatocytes (HFHF diet model)

• Myoblast differentiation processes

• Treatment of melanoma cells with CPI-613 and hydroxychloroquine

This translocation makes PDIA3 accessible for antibody binding, which can be leveraged for both research and potential therapeutic applications .

How are PDIA3 antibodies being explored as potential therapeutic agents in cancer research?

Recent investigations have revealed promising therapeutic applications for PDIA3 antibodies in cancer treatment:

Cancer-specific mechanisms of action:

• The ICT antibody (anti-PDIA3) induces apoptosis in melanoma cells when combined with CPI-613 (a metabolism disruptor) and hydroxychloroquine (HCQ)

• The mechanism appears dependent on PDIA3 expression at the cell surface membrane, which increases after metabolic stress induction

• siRNA-mediated downregulation experiments confirmed that the antibody's effect requires PDIA3 expression

Research findings on cancer specificity:

• PDIA3 antibody therapy shows efficacy in both BRAF wild-type and BRAF-mutated melanoma cell lines

• The approach may be applicable to other malignancies where PDIA3 is expressed on the cell surface, including T-cell leukemia and breast cancer

• Combination therapy (antibody + metabolic disruptors) may overcome resistance mechanisms by targeting stress response pathways

This research direction represents a novel immunotherapeutic approach that selectively targets cancer cells experiencing metabolic stress, potentially offering advantages over conventional therapies .

What methodological approaches are used to investigate PDIA3's interaction with integrin signaling during myogenesis?

PDIA3 plays a crucial role in myoblast differentiation through interactions with β3 integrin. Research methodologies to study this include:

Techniques for investigating PDIA3-integrin interactions:

• Co-immunoprecipitation assays using anti-PDIA3 antibodies to pull down β3 integrin complexes

• Functional blocking studies using both anti-PDIA3 and anti-β3 integrin antibodies to assess pathway dependencies

• Vitronectin stimulation (β3 integrin ligand) combined with PDIA3 blocking to examine signaling pathway integration

Signaling pathway analysis:

• Western blotting for downstream effectors (p-AKT, p-mTOR, myogenin, MyHC) after antibody treatments

• Comparison of membrane-impermeable inhibitors (bacitracin) with antibody-based inhibition to differentiate extracellular vs. intracellular functions

• Temporal analysis of PDIA3 secretion during myoblast differentiation via ELISA

Research has demonstrated that PDIA3-mediated myoblast differentiation requires β3 integrin and depends on the AKT/mTOR pathway. The inhibition of PDIA3 in muscle injuries disrupts myoblast differentiation, impairs muscle regeneration, and ultimately aggravates muscle damage .

What advanced protocols exist for using PDIA3 antibodies in proteomic studies?

PDIA3 antibodies can be integrated into sophisticated proteomic workflows:

Mass spectrometry-coupled immunoprecipitation:

• Peptide mass fingerprinting of immunoprecipitated proteins can identify PDIA3-interacting partners

• Targeted proteomics using parallel reaction monitoring (PRM) can quantify specific PDIA3 peptides in complex samples

• Label-free quantification methods enable comparison of PDIA3 levels across experimental conditions

Protocol considerations:

• Set false discovery rate (FDR) to 5% for database searches

• Incorporate heavy-labeled PDIA3 peptides as internal standards for absolute quantification

• For studying secreted PDIA3 complexes, use heparin-agarose affinity purification followed by immunoblotting

Recent methodological innovations:

• BirA-HA tagging systems for proximity labeling of PDIA3 partners

• Affinity purification using custom oligonucleotide primers for target specificity

• Coupling with XhoI and XbaI digestion for recombinant expression systems

These advanced proteomic approaches have revealed PDIA3's involvement in diverse protein interaction networks and identified novel epitopes recognized by autoantibodies in disease states .

How should researchers address data discrepancies when different PDIA3 antibodies yield contradictory results?

When faced with discrepant results using different PDIA3 antibodies, researchers should implement a systematic troubleshooting approach:

Sources of antibody discrepancies:

• Epitope accessibility differences (conformational vs. linear epitopes)

• Cross-reactivity with related PDI family members

• Clone-specific affinities for different PDIA3 post-translational modifications

• Batch-to-batch variability in commercially produced antibodies

Resolution strategies:

• Perform siRNA knockdown validation with each antibody to confirm specificity

• Compare monoclonal vs. polyclonal antibodies targeting different epitopes

• Test antibodies on recombinant PDIA3 to assess direct binding capacity

• Validate with orthogonal techniques (e.g., mass spectrometry) when possible

Documentation best practices:

• Maintain detailed records of antibody clones, lots, and sources

• Report comprehensive validation data in publications

• Document application-specific optimization parameters

• Consider antibody registry databases for standardization