ERP29 Antibody Pair

What is ERP29 and why is it a significant research target?

ERP29 (Endoplasmic Reticulum Protein 29) is a chaperone protein primarily localized in the endoplasmic reticulum that plays a critical role in protein folding and secretion. It is widely expressed in various tissues and is involved in early protein processing events within the ER .

ERP29 has gained significant research attention due to its:

• Involvement in ER stress responses and protein quality control mechanisms

• Tissue-specific roles in cancer progression

• Participation in multiple signaling pathways including WNT, MAPK, and PI3K/AKT

• Association with drug resistance mechanisms, particularly to cisplatin (CDDP) in cancer

Studies have demonstrated that ERP29 expression varies across different disease states, making it a valuable biomarker and potential therapeutic target in conditions ranging from cancer to age-related degenerative disorders .

When optimizing Western blotting for ERP29 detection, consider these methodological approaches:

• Sample preparation:

  • Use membrane-enriched extracts to increase sensitivity since ERP29 is an ER-resident protein

  • Documented positive control samples include MDCK, NIH/3T3, HepG2, A549, and MCF7 cell lines

• Gel conditions:

  • Use 12% SDS-PAGE gels for optimal separation as ERP29 has a molecular weight of 29 kDa

  • Include molecular weight markers to confirm the correct band size

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membranes (PVDF may provide better signal-to-noise ratio)

  • Block with 5% non-fat milk or BSA in TBST

• Antibody incubation:

  • Primary antibody: Start with 1:1000 dilution (adjust based on specific antibody)

  • Secondary antibody: HRP-conjugated anti-rabbit IgG (most ERP29 antibodies are rabbit polyclonal )

• Detection and troubleshooting:

  • Use enhanced chemiluminescence for visualization

  • If background is high, increase washing steps or decrease antibody concentration

  • For weak signals, consider longer exposure times or signal enhancement systems

Research has shown that ERP29 expression may vary significantly between different cell types and physiological conditions, so optimization may be required for specific experimental contexts .

What experimental controls should be included when validating ERP29 antibody specificity?

When validating ERP29 antibody specificity, both positive and negative controls should be incorporated:

Positive Controls:

• Cell lines with known ERP29 expression: HEK-293, L02, HeLa cells

• Tissue samples: Mouse liver, kidney, lung; rat testis tissue

• Recombinant ERP29 protein as a reference standard

Negative Controls:

• Primary antibody omission control

• Use of non-specific IgG of the same host species

• ERp29-knockout or silenced cells (via siRNA or CRISPR systems)

• Peptide competition assay using the immunizing peptide that was used to generate the antibody

Validation Methods:

• Cross-reactivity assessment: Test reactivity with closely related proteins

• Molecular weight verification: Confirm the observed band is at the expected 29 kDa

• Multiple antibody approach: Use antibodies targeting different epitopes of ERP29

• Orthogonal validation: Corroborate protein expression with mRNA levels

Research has demonstrated that rabbit polyclonal antibodies against ERP29 show cross-reactivity with human, mouse, rat, and other species including dog, cow, sheep, and guinea pig samples , providing versatility for comparative studies across species.

ERP29 functions as an important regulator in the endoplasmic reticulum stress (ERS) response pathway:

• Protective role: ERP29 attenuates ERS-induced apoptosis in various cellular contexts

  • Overexpression of ERP29 increases levels of GRP78, p58IPK, and Nrf-2 (protective proteins)

  • Reduces phosphorylated eIF2α and CHOP (pro-apoptotic markers)

• Temporal dynamics: ERP29 shows distinct expression patterns in response to stress

  • Short-term stress exposure (up to 24 hours): ERP29 levels increase

  • Long-term stress exposure (3 weeks): ERP29 levels decrease

Methods to study ERP29 in ER stress include:

Basic approaches:

• Western blotting to quantify ERP29 and ERS markers (GRP78, CHOP, p-eIF2α)

• RT-qPCR to measure transcript levels

• Immunofluorescence to examine subcellular localization changes

Advanced approaches:

• Proximity ligation assay to detect in situ protein-protein interactions

• CRISPR-Cas9 gene editing to create ERP29-knockout cell lines

• Mass spectrometry to identify ERP29 interacting partners during ERS

• Polysome profiling to examine effects on translation

• Live-cell imaging with fluorescently tagged ERP29

In a study using retinal pigment epithelial (RPE) cells exposed to cigarette smoke extract (CSE), researchers found that overexpression of ERP29 protected cells from CSE-induced apoptosis, while knockdown of ERP29 exacerbated cell death, demonstrating its critical role in stress adaptation .

What strategies can be employed to study ERP29 dimerization and its functional significance?

ERP29 functionality depends on its dimerization state, making this an important aspect to investigate:

Techniques to detect and characterize ERP29 dimerization:

• Chemical cross-linking:

  • Incubate protein samples with cross-linkers (e.g., DSS, DSP)

  • Analyze by SDS-PAGE and immunoblotting with ERP29 antibody

  • Expected observation: ~60 kDa band (dimer) compared to ~30 kDa (monomer)

• Size exclusion chromatography:

  • Separate proteins based on size

  • Compare elution profiles with known standards

  • Western blot fractions to detect ERP29

• Native PAGE:

  • Perform electrophoresis under non-denaturing conditions

  • Western blot to detect native complexes

• Fluorescence techniques:

  • FRET (Förster Resonance Energy Transfer) with fluorescently tagged ERP29

  • Fluorescence Correlation Spectroscopy to determine diffusion rates

Studying functional significance:

• Mutagenesis approaches:

  • Create point mutations at dimerization interface (G37A, L39A, D42A, K52A)

  • Express mutants in cell lines and assess dimerization via cross-linking

  • Evaluate functional outcomes (protein secretion, ER stress responses)

• Domain-based studies:

  • Express N-terminal domain (NTD) or C-terminal domain (CTD) separately

  • Assess dominant-negative effects on wild-type function

  • Perform pull-down assays with His-tagged domains to identify interacting partners

Research has shown that the N-terminal domain of ERP29 is responsible for dimerization, while the C-terminal domain is likely involved in substrate binding. The D42A mutation specifically disrupts dimerization and significantly impairs ERp29 function, highlighting the critical nature of this structural feature .

How can researchers investigate the relationship between ERP29 and microRNA regulation in disease models?

Recent studies have revealed a regulatory feedback loop between ERP29 and microRNAs, particularly miR-135a-5p and miR-4421, which can be studied using these approaches:

Experimental strategies:

• Expression correlation studies:

  • Quantify ERP29 protein levels via Western blot and IHC

  • Measure miRNA expression using qRT-PCR

  • Analyze correlation in clinical samples and cell lines

  • Findings indicate that miR-4421 inhibition increases ERP29 expression

• Direct targeting validation:

  • Luciferase reporter assays with wild-type and mutated ERP29 3'UTR

  • miRNA mimic and inhibitor transfections followed by ERP29 quantification

  • RISC immunoprecipitation to confirm physical interaction

• Feedback mechanism investigation:

  • Manipulate ERP29 expression (overexpression/silencing) and measure miRNA levels

  • ChIP assays to examine effects on promoter methylation

  • Analysis of IL-1β-mediated signaling pathways

• Functional impact assessment:

  • miRNA modulation followed by phenotypic assays

  • Combined manipulation of miRNA and ERP29 to determine epistatic relationships

  • In vivo models testing therapeutic potential of miRNA inhibitors

Research findings:

• In colorectal cancer, miR-135a-5p directly targets ERP29 mRNA

• Conversely, ERP29 suppresses IL-1β-elicited methylation of the miR-135a-5p promoter region

• This creates a homeostatic feedback loop that maintains appropriate levels of both molecules

• In cancer, this balance is disrupted, contributing to disease progression

• Lower ERP29 and higher miR-4421 expressions correlate with poor survival in patients

These findings suggest potential therapeutic applications targeting the ERP29-miRNA regulatory axis in diseases like colorectal and pharyngeal squamous cell carcinomas.

What are the key considerations when designing immunohistochemistry experiments with ERP29 antibodies?

Optimizing immunohistochemistry (IHC) protocols for ERP29 detection requires attention to several critical factors:

Tissue preparation and fixation:

• Formalin-fixed paraffin-embedded (FFPE) sections typically yield good results

• Optimal section thickness: 4-6 μm

• Freshly cut sections are preferable to stored slides

Antigen retrieval:

• Heat-induced epitope retrieval (HIER) is recommended

• Two effective buffer options:

  • TE buffer pH 9.0 (preferred)

  • Citrate buffer pH 6.0 (alternative)

• Microwave or pressure cooker for 10-20 minutes

Antibody selection and optimization:

• Rabbit polyclonal antibodies show good reactivity across multiple species

• Recommended dilution range: 1:50-1:500

• Incubation conditions: 4°C overnight or 1-2 hours at room temperature

• Both DAB (chromogenic) and fluorescence detection systems work well

Controls and validation:

• Positive tissue controls: Human liver cancer, breast cancer, pancreas; mouse liver, kidney

• Negative controls: Primary antibody omission, isotype control

• Counterstain: Hematoxylin for nuclear visualization

• Consider dual staining with ER markers (calnexin, PDI) for co-localization

Analysis considerations:

• ERP29 shows primarily cytoplasmic/ER localization pattern

• Expression levels vary significantly between cell types within the same tissue

• ERp29 expression profile largely parallels that of protein disulfide isomerase (PDI) but with strikingly different ERp29/PDI ratios in various cell types

Research has identified 35 functionally distinct cell types in rat tissues that express ERP29, establishing it as a general ER marker but with tissue-specific expression patterns that may correlate with secretory activity .

How can ERP29 antibodies be used to study the effects of cisplatin resistance in cancer models?

ERP29 has emerged as an important factor in cisplatin (CDDP) resistance mechanisms, particularly in pharyngeal squamous cell carcinoma (PSCC). Researchers can investigate this relationship using these methodological approaches:

Cell model establishment:

• Develop cisplatin-sensitive, cisplatin-treated, and cisplatin-resistant cell lines:

  • CDDP-sensitive cells (e.g., FaDu, LAU-2063)

  • CDDP-treated cells (e.g., FaDu-CDDP)

  • CDDP-resistant cells (e.g., FaDu-R)

• Verify resistance phenotype through dose-response curves and IC50 determination

ERP29 expression analysis:

• Quantify baseline ERP29 expression across cell models using:

  • Western blotting (protein level)

  • qRT-PCR (mRNA level)

  • Flow cytometry (per-cell analysis)

• Key finding: FaDu-R cells show decreased ERP29 expression compared to sensitive and treated cells

Functional studies:

• Modify ERP29 expression:

  • Overexpression using expression vectors

  • Silencing using siRNA or shRNA

• Assess cellular behaviors:

  • Cell proliferation and cell cycle analysis

  • Necrosis and apoptosis detection

  • Migration assays

  • 3D spheroid formation with E-cadherin/vimentin assessment

• Evaluate cisplatin sensitivity changes through:

  • MTT/MTS viability assays at varying drug concentrations

  • Colony formation assays

  • Apoptosis markers (Annexin V, PARP cleavage)

Mechanistic investigations:

• Gene expression analysis:

  • PCR arrays for WNT, MAPK, and PI3K/AKT pathway components

  • Validation by qPCR for key genes (SOS1, MAPK1, AKT1, CASP9)

• Protein interaction studies:

  • Co-immunoprecipitation with ERP29 antibodies

  • Immunofluorescence co-localization

miRNA-based approaches:

• Assess miR-4421 effects:

  • Transfect miR-4421 inhibitors

  • Measure changes in ERP29 expression

  • Quantify expression of target genes (MAPK1, AKT1, JUN)

• Survival correlation analysis in patient samples

Recent research found that ERP29 silencing decreases necrotic cell death and increases migration in cisplatin-sensitive, treated, and resistant cells. During cisplatin treatment, ERP29 silencing enhances cell proliferation and alters expression of genes involved in key signaling pathways, suggesting its important role in drug resistance mechanisms .

What are the advanced considerations for using ERP29 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) using ERP29 antibodies can reveal novel protein interactions and complexes, but requires careful optimization:

Experimental design considerations:

• Lysis buffer optimization:

  • Use mild non-denaturing buffers to preserve protein-protein interactions

  • Include appropriate protease/phosphatase inhibitors

  • Consider specialized buffers for ER proteins (e.g., containing low concentrations of detergents like digitonin or NP-40)

• Antibody selection:

  • Choose antibodies validated for IP applications

  • Consider epitope accessibility in native protein complexes

  • Amount needed: 0.5-4.0 μg antibody per 1-3 mg of protein lysate

  • Pre-clear lysates to reduce non-specific binding

• Controls to include:

  • Input control (5-10% of starting material)

  • IgG control (same species/amount as ERP29 antibody)

  • Reciprocal IP (IP with antibodies against suspected interacting proteins)

  • Negative control samples (ERP29-knockdown cells)

• Detection strategies:

  • Western blot with antibodies against potential interacting partners

  • Mass spectrometry for unbiased identification of protein complexes

  • Functional validation of identified interactions

Known ERP29 interactions to investigate:

• Calnexin and Grp94 (ER chaperones)

• PDI family members (protein folding)

• Components of WNT, MAPK, and PI3K/AKT pathways

• miRNA processing machinery

Advanced technical considerations:

• Crosslinking approaches:

  • Reversible crosslinkers can stabilize transient interactions

  • Optimized protocols show ERP29 homodimers at ~60 kDa

• Subcellular fractionation:

  • Enrich for ER fractions before IP to increase specificity

  • Enables detection of compartment-specific interactions

• Proximity-dependent labeling:

  • BioID or APEX2 fusions with ERP29 for in vivo proximity labeling

  • Identifies spatial protein networks around ERP29

Research has demonstrated that ERP29 forms homodimers that are essential for its function, and that the N-terminal domain is responsible for this dimerization. Co-IP experiments have successfully identified interactions between ERP29 and other ER proteins involved in protein folding and secretion .

How can researchers investigate the role of ERP29 in epithelial-mesenchymal transition (EMT) in cancer models?

ERP29's involvement in epithelial-mesenchymal transition (EMT) can be studied through these methodological approaches:

Cellular models and markers:

• 3D spheroid cultures:

  • Establish 3D spheroid cultures from cancer cell lines

  • Modify ERP29 expression (overexpression/silencing)

  • Assess key findings:

    • ERP29 silencing decreases E-cadherin expression

    • ERP29 silencing increases vimentin expression

• EMT marker profiling:

  • Western blot/IF analysis of canonical markers:

    • Epithelial: E-cadherin, ZO-1, PAR3

    • Mesenchymal: Vimentin, N-cadherin, Fibronectin

  • qRT-PCR for transcriptional regulators (SNAIL, SLUG, ZEB1/2, TWIST)

Functional assays:

• Migration and invasion:

  • Scratch/wound healing assays

  • Transwell migration/invasion assays

  • Time-lapse microscopy

  • Results indicate ERP29 silencing increases migration in CDDP-sensitive, treated, and resistant cells

• Cell adhesion:

  • Matrix adhesion assays

  • Cell-cell aggregation assays

  • E-cadherin function assays (calcium switch)

• Morphological analysis:

  • Phase-contrast microscopy for morphological changes

  • Cytoskeletal reorganization (F-actin staining)

Molecular mechanisms:

• Signal pathway analysis:

  • WNT pathway activation (β-catenin localization)

  • MAPK pathway (ERK1/2 phosphorylation)

  • PI3K/AKT signaling (AKT phosphorylation)

  • TGF-β signaling (SMAD activation)

• Gene expression regulation:

  • ChIP assays for promoter binding

  • 3'UTR reporter assays for miRNA regulation

  • RNA-seq for global expression changes

Translational approach:

• Clinical correlations:

  • IHC analysis of patient samples for ERP29 and EMT markers

  • Correlation with prognostic indicators and survival outcomes

  • Metastasis association analysis