ecrg4b Antibody

What is ECRG4/ecrg4b and why is it important to study with antibody-based approaches?

ECRG4 (Esophageal Cancer-Related Gene 4), also known as Augurin or C2orf40 in humans and ecrg4b in zebrafish, is a 148 amino acid secreted protein that functions as a probable hormone. ECRG4 attenuates cell proliferation and induces senescence of oligodendrocyte and neural precursor cells in the central nervous system . ECRG4-induced senescence is characterized by G1 arrest, RB1 dephosphorylation, and accelerated CCND1 and CCND3 proteasomal degradation .

Antibody-based approaches are essential for studying ECRG4/ecrg4b because:

• ECRG4 has been suggested to act as a tumor suppressor, making it valuable for cancer research

• There is a discrepancy between detectable mRNA and protein levels, requiring protein-level validation

• Post-translational modifications and processing of ECRG4 can be studied using antibodies

• Antibodies allow localization studies in tissue context through immunohistochemistry and immunofluorescence

Zebrafish ecrg4b specifically has been identified as a target of the Yap/Taz signaling pathway and is expressed in the presumptive epidermis during development, making it valuable for developmental biology research .

What applications are validated for commercial ECRG4/ecrg4b antibodies?

Based on available data, commercial ECRG4 antibodies have been validated for the following applications:

When designing experiments, researchers should note that antibody validation remains a challenge in the field . Independent validation using positive and negative controls is strongly recommended before proceeding with critical experiments.

How can I validate the specificity of an ECRG4/ecrg4b antibody for my research?

Rigorous validation of ECRG4/ecrg4b antibodies is essential, particularly given the documented issues with antibody specificity in other research areas . A comprehensive validation approach should include:

• Positive and negative cell line controls: Use cell lines with confirmed ECRG4 expression (via RNA-seq or qPCR) as positive controls and those lacking expression as negative controls. The HCT116 and T47D cell lines have been confirmed to lack ECRG4 mRNA expression (<1 FPKM) and can serve as negative controls .

• Genetic manipulation controls: Compare cells engineered to overexpress ECRG4 with their parental counterparts. For example, lentivirus-engineered expression of FLAG-tagged ECRG4 can create reliable positive controls .

• Multiple antibody-based techniques: Validate using complementary methods:

  • Western blot: Should show a single band at ~17 kDa (or ~120 kDa for fusions)

  • Immunofluorescence: Should show expected subcellular localization

  • Flow cytometry: Should show surface expression for secreted ECRG4

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding. This approach was successfully used to map epitopes in the EphB4 cysteine-rich region .

• Immunoprecipitation with mass spectrometry: Identify bound proteins by IP followed by MS to confirm that the antibody is capturing the intended target .

• Parallel mRNA quantification: Compare antibody-based protein detection with mRNA levels measured by qPCR or RNA-seq to ensure correlation .

The most rigorous validation would employ knockout or knockdown models where ECRG4/ecrg4b expression is eliminated or significantly reduced.

What are the key considerations for using ecrg4b antibodies in zebrafish research models?

When using antibodies to study ecrg4b in zebrafish models, researchers should consider:

• Developmental expression patterns: ecrg4b is expressed in the presumptive epidermis of zebrafish embryos at the 18-somite stage, but is not detected in yap1;wwtr1 double mutants . This spatiotemporal pattern must be considered when designing experiments.

• Antibody cross-reactivity: Ensure the antibody recognizes zebrafish ecrg4b specifically. Many commercial antibodies are raised against human ECRG4 and may have limited cross-reactivity with zebrafish orthologs.

• Controls for transgenic models: When using transgenic zebrafish, appropriate controls must be included:

  • For heat-shock inducible systems (like HS:DN-yap or HS:CA-yap), include non-transgenic siblings as controls

  • Confirm genotypes by PCR after in situ hybridization

• Sample preparation for antibody applications:

  • For immunohistochemistry in zebrafish larvae: Permeabilize with ice-cold acetone at -20°C for 5 min, wash in H2O for 5 min, followed by 5 × 5 min washes in PBS, and block overnight with PBS containing 2% goat serum and 1% bovine serum albumin (BSA)

  • For flow cytometry: Dissociate larvae in cold trypsin-EDTA solution by trituration, halt dissociation with HBSS supplemented with 10% fetal bovine serum and 100 μg/mL DNaseI, filter through cell strainers, and process for flow sorting

• RNA analysis correlations: Complement antibody-based studies with RNA analysis techniques like in situ hybridization with FISH probes to verify expression patterns .

• Model validation: Verify that manipulations of Yap/Taz signaling affect ecrg4b expression as expected. For example, expression of DN-yap decreases ecrg4b expression relative to controls, whereas CA-yap enhances ecrg4b expression .

How can I optimize Western blot protocols for detecting ECRG4/ecrg4b?

Optimizing Western blot protocols for ECRG4/ecrg4b detection requires attention to several key parameters:

• Antibody concentration: Start with the manufacturer's recommended dilution. For example:

  • Anti-C2orf40/ECRG4 antibody (ab224077) has been used at 1/100 dilution

  • ECRG4 Polyclonal Antibody (bs-9807R) is recommended at 1:300-5000 dilution

  • Anti-ECRG4 antibody (SAB4503353) is typically used at 1:20000 for ELISA

• Positive controls: Include a positive control such as:

  • Cell lysate from cells transfected with ECRG4 expression vector

  • Recombinant ECRG4 protein

  • Tissues known to express high levels of ECRG4 (testis, ovary, placenta)

• Sample preparation:

  • Use appropriate lysis buffers that maintain protein integrity

  • Include protease inhibitors to prevent protein degradation

  • For secreted forms, consider analyzing both cell lysates and conditioned media

• Expected band size:

  • Native ECRG4: ~17 kDa

  • Tagged versions: adjust for tag size (e.g., C-terminal myc-DDK tag adds ~3.1 kDa)

  • Check for potential glycosylation or other post-translational modifications

• Membrane transfer conditions:

  • For this small protein, use PVDF membrane with 0.2 μm pore size

  • Consider using wet transfer at lower voltage for longer time

• Troubleshooting strategies:

  • If no signal: Increase antibody concentration, extend incubation time, use enhanced detection reagents

  • If high background: Increase blocking time, decrease antibody concentration, use more stringent washing

  • If multiple bands: Validate specificity with knockout/knockdown controls

The Western blot image shown in search result demonstrates specific detection of ECRG4 when comparing negative control (vector-only transfected HEK-293T) with ECRG4 overexpression cells.

How do ECRG4/ecrg4b expression patterns differ across tissue types and disease states?

ECRG4/ecrg4b expression varies significantly across tissues and is altered in various disease states, particularly cancer:

Normal Tissue Expression Patterns:

• Human ECRG4 protein is consistently detected in testis, ovary, and placenta (weak expression)

• ECRG4 is expressed in lymphoid cells

• In zebrafish, ecrg4b is expressed in the presumptive epidermis during development

• ECRG4 is found in the central nervous system where it may function as a neuronal peptide hormone

Disease-Associated Expression Patterns:

• ECRG4 was originally identified as a candidate tumor suppressor in esophageal cancer

• Decreased expression has been observed in various cancers, consistent with its potential tumor suppressor role

• Protein is detected in granuloma cell tumors and a subset of malignant melanoma and thyroid cancers

• ECRG4 functions as a proinflammatory factor in macrophages/microglia and may play a role in immune responses

Experimental Models:

• In zebrafish models, ecrg4b expression is regulated by the Yap/Taz signaling pathway:

  • Expression decreases in yap1;wwtr1 double mutant embryos

  • Decreased expression observed with DN-yap (dominant negative)

  • Enhanced expression with CA-yap (constitutively active)

Understanding these expression patterns is crucial when selecting appropriate experimental models and interpreting results. When studying ECRG4 in disease states, researchers should use multiple approaches to confirm expression changes, including mRNA quantification (qPCR, RNA-seq) alongside antibody-based protein detection methods.

How can contradictory results between mRNA and protein expression of ECRG4/ecrg4b be reconciled?

Contradictory results between mRNA and protein expression of ECRG4/ecrg4b are a documented challenge in the field. Research has shown that "most cell lines have been reported to lack ERβ mRNA... while antibody-based applications report its protein expression" . Although this citation specifically refers to ERβ research, similar discrepancies apply to ECRG4 research.

To reconcile these contradictions:

• Validate antibody specificity thoroughly:

  • Confirm antibody specificity using positive and negative controls

  • Use cells with confirmed ECRG4 mRNA absence as negative controls

  • Include genetically modified cells (overexpression, knockout) as reference points

• Consider post-transcriptional regulation:

  • ECRG4 may be subject to microRNA regulation or RNA stability factors

  • Examine half-life of the ECRG4 mRNA versus protein

• Investigate protein secretion and processing:

  • ECRG4 is a secreted protein, so intracellular levels may not correlate with expression

  • Check both cell lysates and conditioned media

  • Consider that different antibodies may recognize distinct processed forms

• Use complementary techniques:

  • Combine RNA-seq, qPCR, Western blot, immunohistochemistry, and ELISA

  • RT-qPCR from RNA immunoprecipitation can help quantify expression in specific cell types

  • Use in situ hybridization alongside immunostaining

• Implement statistical approaches:

  • Perform correlation analyses between mRNA and protein levels across multiple samples

  • Use multiple reference genes/proteins for normalization

• Consider technical limitations:

  • Sensitivity differences between mRNA and protein detection methods

  • The arbitrary thresholds used to define "positive" or "negative" expression

When reporting contradictory results, transparently describe all methods, controls, and limitations to help advance understanding of these discrepancies in the field.

What are the emerging techniques for studying ECRG4's interactions with scavenger receptors?

Recent research has identified that ECRG4, particularly amino acid residues 71-132 of ECRG4 (ECRG4(71-132)), binds to multiple scavenger receptors, including lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), Scarf1, Cd36, and Stabilin-1 . These interactions appear important for the protein's proinflammatory functions. Emerging techniques to study these interactions include:

• Retrovirus-mediated expression cloning:

  • This approach successfully identified LOX-1 as a membrane protein that binds ECRG4(71-132)

  • Can be applied to identify additional potential receptors

• Competitive inhibition assays:

  • Polyinosinic acid, a broad competitive inhibitor of scavenger receptors, can be used to assess binding specificity

  • This approach demonstrated reduced binding of ECRG4(71-132) and diminished NF-κB activation in microglia

• Cell encapsulation systems:

  • Co-encapsulation of cells in microdroplets allows for high-throughput screening of receptor-ligand interactions

  • Similar approaches using primary B cells and reporter cells in agarose-based microdroplets have been used for agonist antibody discovery

• Peptide mapping and competition assays:

  • Designing overlapping peptides corresponding to different regions of ECRG4

  • Using these peptides in competition assays to identify specific binding domains

  • This approach proved successful in mapping antibody epitopes for EphB4

• Proximity-based labeling techniques:

  • BioID or APEX2-based proximity labeling to identify proteins in close proximity to ECRG4 in living cells

  • Can reveal transient interactions with receptors

• Advanced microscopy approaches:

  • Super-resolution microscopy to visualize co-localization of ECRG4 with scavenger receptors

  • Fluorescence resonance energy transfer (FRET) to confirm direct protein-protein interactions

• MyD88-dependent signaling analysis:

  • Since ECRG4 signal transduction involves MyD88, an adaptor protein for Toll-like receptors (TLRs)

  • Analysis of downstream signaling can help characterize receptor engagement

These advanced techniques can help elucidate the molecular mechanisms by which ECRG4 interacts with scavenger receptors and exerts its biological functions in inflammation and other processes.

What are the best practices for optimizing immunohistochemistry protocols for ECRG4/ecrg4b detection?

Optimizing immunohistochemistry (IHC) protocols for detecting ECRG4/ecrg4b requires careful attention to several parameters:

• Antibody selection and validation:

  • Several antibodies are validated for IHC-P, including ab224077 and bs-9807R

  • Confirm specificity using positive and negative controls before proceeding

  • Consider antibodies targeting different epitopes to confirm results

• Sample preparation:

  • Fixation: 10% neutral buffered formalin is standard, but optimize fixation time

  • Antigen retrieval: Test both heat-induced epitope retrieval (HIER) and enzymatic methods

  • For HIER, compare citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

• Blocking and antibody incubation:

  • Thorough blocking is essential: use 2-5% normal serum from the same species as the secondary antibody

  • For zebrafish larvae: block overnight with PBS containing 2% goat serum and 1% BSA

  • Antibody dilution ranges:

    • bs-9807R: 1:200-400 for IHC-P

    • OAPB01487: start with manufacturer recommendations and titrate

• Detection systems:

  • For low expression: use amplification systems like tyramide signal amplification

  • For co-localization studies: use fluorescent secondary antibodies (IF-IHC)

  • For quantitative analysis: use chromogenic detection with controlled development times

• Controls to include:

  • Positive tissue controls: Testis, ovary, and placenta show ECRG4 expression

  • Negative tissue controls: Tissues known to lack ECRG4 expression

  • Technical controls: Omit primary antibody; use isotype control antibody

• Optimization strategies:

  • Use a matrix approach testing different antibody concentrations and antigen retrieval methods

  • Document all optimization steps for reproducibility

  • Consider multiplex staining to co-localize with known markers

• Quantification approaches:

  • Define clear scoring criteria before analysis

  • Use digital image analysis software for unbiased quantification

  • Consider both staining intensity and percentage of positive cells

When publishing results, include detailed methods sections describing all optimization steps, antibody validation, and controls used to enable reproducibility by other researchers.

How can computational approaches improve ECRG4/ecrg4b antibody design and specificity?

Computational approaches offer powerful tools for improving antibody design and specificity for ECRG4/ecrg4b research:

• Epitope prediction and optimization:

  • Computational algorithms can identify potential antigenic regions of ECRG4

  • Tools like BepiPred, DiscoTope, and PEPOP can predict B-cell epitopes

  • These predictions can guide selection of immunogens for antibody production

• Structure-based antibody design:

  • If structural data for ECRG4 is available, molecular modeling can identify accessible epitopes

  • RosettaAntibodyDesign (RAbD) can be used to design antibodies with improved binding properties

  • Though computational protein design is challenging, tools like RAbD can be trusted "with qualifications"

• Cross-reactivity prediction:

  • Sequence comparison across species can identify conserved vs. variable regions

  • This helps design antibodies that are either species-specific or cross-reactive

  • BLAST analysis of potential epitopes against the proteome can identify sequences that might cause cross-reactivity

• Antibody engineering optimization:

  • Computational approaches can identify mutations to improve:

    • Binding affinity

    • Specificity

    • Stability

    • Solubility

• Validation strategies guided by computation:

  • Identify structurally similar proteins that might cross-react

  • Design competitive peptides for validation experiments

  • Generate recombinant protein fragments for validation

• Practical considerations for computational antibody design:

  • "How many designs did your colleague make and order? [...] even for redesign, you still want around 20 designs per project (at least)"

  • "Were the computations run on a large cluster? How many decoys were created?"

  • For de-novo design, consider complementing with display technologies: "If you are serious about de-novo design, I would also think about getting a yeast display platform up and running"

Computational approaches should be used in conjunction with experimental validation, as the field acknowledges that "computational protein design is hard" and results require experimental confirmation.

What controls should be included when studying ECRG4/ecrg4b's role in cellular senescence?

ECRG4 has been implicated in inducing senescence of oligodendrocyte and neural precursor cells . When studying this function, appropriate controls are essential:

• Cellular model controls:

  • Positive controls: Cells with verified ECRG4 expression

  • Negative controls: ECRG4 knockout or knockdown cells

  • Overexpression controls: Cells with induced ECRG4 expression under controllable promoters

  • Pathway controls: Cells with manipulated RB1 phosphorylation status

• Molecular signaling controls:

  • Monitor CCND1 and CCND3 proteasomal degradation, as ECRG4-induced senescence accelerates this process

  • Include RB1 phosphorylation status analysis, as dephosphorylation is characteristic of ECRG4-induced senescence

  • Assess cell cycle arrest at G1 phase using flow cytometry

• Functional assay controls:

  • Senescence markers: Include β-galactosidase staining as a standard senescence marker

  • Proliferation assays: Compare proliferation rates between ECRG4-expressing and non-expressing cells

  • Cell cycle analysis: Include full cell cycle profiling to confirm G1 arrest

• Antibody specificity controls:

  • Validate all antibodies used (anti-ECRG4, anti-RB1, anti-CCND1, anti-CCND3)

  • Include isotype controls and secondary-only controls for immunofluorescence

  • Use peptide competition assays to confirm antibody specificity

• Manipulation verification controls:

  • Confirm ECRG4 overexpression or knockdown at both mRNA and protein levels

  • For inducible systems, include non-induced controls

  • For zebrafish models, verify that manipulations of Yap/Taz signaling affect ecrg4b expression as expected

• Experimental timeline controls:

  • Include multiple time points to capture the dynamic process of senescence induction

  • For long-term studies, maintain parallel cultures to assess stability of phenotype

• Statistical controls:

  • Use appropriate statistical tests based on data distribution

  • Include sufficient biological and technical replicates

  • Calculate sample sizes needed for adequate statistical power

By including these comprehensive controls, researchers can more confidently attribute observed senescence phenotypes to ECRG4/ecrg4b function rather than experimental artifacts or confounding factors.

How should researchers interpret differences in results across various antibody-based techniques for ECRG4/ecrg4b?

Different antibody-based techniques may yield varying results when studying ECRG4/ecrg4b. Understanding and interpreting these differences requires careful consideration:

• Technique-specific considerations:

  Technique	Sensitivity	Specificity Concerns	Best For
  Western blot	Moderate	Size-based discrimination helps	Protein size verification
  IHC/IF	High	Cross-reactivity with fixed tissues	Spatial localization
  ELISA	Very high	Hidden epitopes in native proteins	Quantification
  Flow cytometry	Moderate-high	Surface vs. intracellular distinction	Cell-specific expression

• Reconciling contradictory results:

  • When techniques disagree, consider that each detects different aspects of the protein

  • Western blot assesses denatured protein size but may miss post-translational modifications

  • IHC preserves tissue architecture but may have fixation artifacts

  • ELISA is highly quantitative but removes cellular context

• Antibody-specific factors:

  • Different antibodies recognize different epitopes, which may be:

    • Differentially accessible in various techniques

    • Affected differently by fixation or denaturation

    • Subject to masking by protein-protein interactions

  • As observed with ERβ antibodies, "results generated with the three ERβ antibodies using IHC and WB are not congruent"

• Biological explanations for discrepancies:

  • Protein processing: ECRG4 undergoes proteolytic processing

  • Subcellular localization: As a secreted protein, ECRG4 distribution varies

  • Post-translational modifications may affect epitope recognition

• Methodological approach to discrepancies:

  • Verify results using multiple antibodies targeting different epitopes

  • Complement antibody techniques with non-antibody methods (e.g., mass spectrometry)

  • Use genetic approaches (overexpression, knockout) to validate findings

  • For critical findings, employ orthogonal techniques that don't rely on antibodies

• Documentation and reporting:

  • Transparently report discrepancies between techniques

  • Document detailed protocols to enable others to reproduce conditions

  • Include all relevant controls for each technique

  • Consider publishing negative results to advance the field