ERVFRD-1 Antibody, Biotin conjugated

What is ERVFRD-1 and what cellular processes is it involved in?

ERVFRD-1 (endogenous retrovirus group FRD member 1) encodes syncytin-2, a 538 amino acid protein with a molecular weight of approximately 59.5 kDa. It belongs to the Gamma type-C retroviral envelope protein family and is primarily localized in the cell membrane . The protein undergoes several post-translational modifications, including protein cleavage and glycosylation.

Functionally, ERVFRD-1 plays crucial roles in:

• Trophoblast cell fusion during placental development

• Regulation of cell cycle in the syncytiotrophoblast

• Immunomodulation within various tissue contexts

The protein is expressed predominantly in placental tissue, with expression levels approximately 10-fold lower than ERVW-1 (syncytin-1) in first-trimester placentas. Interestingly, ERVFRD-1 transcript levels decrease progressively throughout pregnancy, contrasting with ERVW-1 expression patterns .

What are optimal conditions for using biotin-conjugated ERVFRD-1 antibodies in Western blotting?

For optimal Western blotting using biotin-conjugated ERVFRD-1 antibodies, researchers should consider the following protocol:

• Sample preparation:

  • Prepare tissue/cell lysates in RIPA buffer supplemented with protease inhibitors

  • For placental samples, processing should account for high protein content and potential degradation

• Electrophoresis and transfer parameters:

  • Separate proteins on 10-12% SDS-PAGE gels (optimal for 59.5 kDa target)

  • Use wet transfer at 30V overnight at 4°C for complete transfer of glycosylated proteins

• Blocking and antibody incubation:

  • Block membranes with 5% BSA in TBST (preferred over milk-based blockers)

  • Incubate with biotin-conjugated ERVFRD-1 antibody at 1:1000 dilution for 2 hours at room temperature or overnight at 4°C

  • Use streptavidin-HRP (1:5000) for detection

• Detection considerations:

  • Account for potential multiple bands due to protein cleavage and glycosylation

  • Expected primary band at approximately 59.5 kDa with potential secondary bands due to post-translational modifications

How can researchers validate ERVFRD-1 antibody specificity?

Validating antibody specificity is crucial for ensuring reliable experimental results. For ERVFRD-1 antibody validation:

• Positive controls:

  • Use placental tissue samples (especially first-trimester samples) which have confirmed high expression of ERVFRD-1

  • Include cell lines with known ERVFRD-1 expression profiles

• Negative controls:

  • Include tissues with minimal ERVFRD-1 expression

  • Use knockdown/knockout models where available

  • Test isotype control antibodies to confirm binding specificity

• Peptide competition assays:

  • Pre-incubate antibody with excess ERVFRD-1 peptide

  • Compare signal between blocked and unblocked antibody samples

• Cross-reactivity assessment:

  • Test against related proteins, particularly syncytin-1 (ERVW-1), to ensure specificity

  • Confirm signal absence in species lacking ERVFRD-1 orthologs (except mouse and chimpanzee, which have reported orthologs)

What are the expected expression patterns of ERVFRD-1 across normal and pathological tissues?

ERVFRD-1 expression shows distinct patterns across tissues and disease states:

Normal tissue expression:

• Highest expression in placental tissue, particularly in early pregnancy

• Expression decreases progressively throughout gestation

• Low baseline expression in most non-placental tissues

Pathological expression patterns:

• Preeclampsia (PE): Significantly altered expression compared to normal placentas, with greater reduction than observed for ERVW-1

• Kidney renal clear cell carcinoma (KIRC): Generally low expression in tumor tissue compared to normal kidney tissue (P < 0.001)

Expression correlation with clinical parameters in KIRC:

What considerations should be made when extracting and preserving samples for ERVFRD-1 detection?

Optimal sample handling is critical for reliable ERVFRD-1 detection:

• Sample collection:

  • For placental samples: collect from multiple sites to account for expression heterogeneity

  • For tumor samples: include paired normal tissue whenever possible

  • Flash-freeze tissues in liquid nitrogen within 30 minutes of collection

• RNA extraction considerations:

  • Use RNase-free reagents and environment

  • Include DNase treatment to prevent genomic DNA contamination

  • Verify RNA integrity (RIN > 7) before proceeding to expression analysis

• Protein extraction optimization:

  • For membrane-bound proteins like ERVFRD-1, include detergents suitable for membrane protein solubilization

  • Consider subcellular fractionation to enrich for membrane proteins

  • Add phosphatase inhibitors if studying regulatory pathways

• Storage recommendations:

  • Store RNA in RNase-free water at -80°C with minimal freeze-thaw cycles

  • Aliquot protein lysates to avoid repeated freeze-thaw cycles

  • For long-term storage of tissues, consider OCT embedding for future immunohistochemistry

How can ERVFRD-1 antibodies be used to investigate placental pathologies?

Biotin-conjugated ERVFRD-1 antibodies provide valuable tools for investigating placental pathologies:

• Preeclampsia research applications:

  • Immunohistochemistry for localization patterns: preeclamptic placentas show altered ERVFRD-1 localization

  • Quantitative analysis of expression levels using Western blotting or qPCR

  • Correlation with severity: ERVFRD-1 expression levels correlate with PE severity

• Genetic association studies:

  • The rs9393931 variant of ERVFRD-1 shows significant association with PE development

  • Homozygous TT genotype significantly increases PE risk

  • The variant, located in the 3'-UTR region, may affect mRNA processing, stability, and translation efficiency

• Cell fusion investigation methods:

  • Trophoblast fusion assays to assess impact of ERVFRD-1 variants on syncytialization capacity

  • Co-culture systems to evaluate cell-cell fusion dynamics

  • Live cell imaging to track fusion events in relation to ERVFRD-1 expression

• Experimental approaches to study regulation:

  • ChIP assays to investigate GCM1 and p21 binding to ERVFRD-1 promoter

  • Luciferase reporter assays to study transcriptional regulation

  • CRISPR-Cas9 genome editing to study functional consequences of ERVFRD-1 variants

What techniques are recommended for studying ERVFRD-1's role in immune regulation?

Based on recent research showing ERVFRD-1's involvement in immune regulation, particularly in cancer contexts:

• Immune infiltration analysis:

  • Single-sample Gene Set Enrichment Analysis (ssGSEA) to evaluate immune cell enrichment scores

  • Flow cytometry to quantify specific immune cell populations in relation to ERVFRD-1 expression

  • Multiplex immunohistochemistry to assess spatial relationships between ERVFRD-1-expressing cells and immune infiltrates

• Correlation analysis methods:

  • ERVFRD-1 expression shows significant positive correlation with mast cells (r=0.377, P < 0.001)

  • Negative correlation with regulatory T cells (Treg cells) (r = -0.253, P < 0.001)

  • Spearman's correlation analysis for assessing relationships between ERVFRD-1 and immune cell markers

• Functional assays for immune modulation:

  • Co-culture experiments with immune cells and ERVFRD-1-expressing cells

  • Cytokine profiling in relation to ERVFRD-1 expression levels

  • Gene knockdown/overexpression studies to assess direct immune regulatory effects

• Checkpoint inhibitor response prediction:

  • Analysis of relationship between ERVFRD-1 expression and immunotherapy response

  • Correlation with established immune checkpoints (PD-L1/PD-1)

  • Assessment of tumor mutation burden (TMB) in relation to ERVFRD-1 expression

How can researchers effectively study ERVFRD-1 methylation status?

Methylation analysis of ERVFRD-1 requires specific methodological considerations:

• Bisulfite sequencing approach:

  • Design primers specific to bisulfite-converted ERVFRD-1 promoter sequences

  • Account for repetitive nature of retroviral sequences in primer design

  • Include appropriate controls for conversion efficiency

• Methylation-specific PCR optimization:

  • Design primers for methylated and unmethylated versions of the ERVFRD-1 promoter

  • Validate with known methylated and unmethylated controls

  • Optimize annealing temperatures for specificity

• Genome-wide methylation analysis integration:

  • Analyze ERVFRD-1 methylation in the context of genome-wide methylation data

  • Correlate with expression data to establish functional relationships

  • In KIRC specifically, assess relationship between hypomethylation and expression

• Experimental manipulation of methylation:

  • Treatment with DNA methyltransferase inhibitors (e.g., decitabine) to assess impact on ERVFRD-1 expression

  • Correlation with immune response markers after demethylation

  • Potential therapeutic applications in cancer immunotherapy contexts

How can ERVFRD-1 expression analysis contribute to cancer prognosis and treatment?

ERVFRD-1 shows significant potential as a prognostic biomarker, particularly in kidney cancer:

• Prognostic value assessment methods:

  • Kaplan-Meier survival analysis comparing high vs. low ERVFRD-1 expression groups

  • Cox regression analysis incorporating ERVFRD-1 with established clinical variables

  • Nomogram construction for survival prediction

• Predictive nomogram development:

  • A nomogram incorporating ERVFRD-1 expression, pathologic T stage, and age shows promising predictive power for KIRC patient survival

  • The model demonstrates significant value for risk stratification in clinical contexts

• Immunotherapy response prediction:

  • Analysis of correlation between ERVFRD-1 expression and immune markers

  • Higher ERVFRD-1 expression correlates positively with immunostimulators (e.g., CD70, TNFSF14)

  • Lower ERVFRD-1 expression correlates with higher TMB scores, potentially indicating better response to immunotherapy

• Therapeutic target exploration:

  • Assessment of ERVFRD-1 as a direct therapeutic target

  • Investigation of methylation-modifying agents to regulate ERVFRD-1 expression

  • Correlation with response to immune checkpoint blockade therapies

What considerations are important when designing functional studies to differentiate ERVFRD-1 from related proteins?

Differentiating ERVFRD-1 (syncytin-2) from related proteins, particularly syncytin-1 (ERVW-1):

• Experimental design considerations:

  • Account for different expression patterns: ERVFRD-1 shows higher expression in early pregnancy with progressive decrease, while ERVW-1 shows different temporal patterns

  • Design knockdown experiments with highly specific siRNAs to avoid cross-reactivity

  • Include both proteins in experimental analyses to determine specific contributions

• Functional domain analysis:

  • Target unique domains for specific antibody binding

  • Use domain-specific blocking peptides for functional studies

  • Design mutational studies focusing on non-conserved regions

• Cell type specificity:

  • Leverage differential expression across cell types for functional studies

  • Design cell type-specific knockout models

  • Use conditional expression systems to control timing of expression

• Technical approaches for differentiation:

  • Employ super-resolution microscopy for co-localization studies

  • Use proximity ligation assays to study protein-protein interactions

  • Design dual-color imaging techniques to simultaneously track both proteins

What are common issues in ERVFRD-1 detection and how can they be resolved?

Researchers frequently encounter specific challenges when working with ERVFRD-1 antibodies:

• Sensitivity issues:

  • Problem: Low signal in tissues with expected expression

  • Solution: Implement signal amplification methods such as tyramide signal amplification

  • Optimization: Test multiple antibody concentrations and incubation times

• Specificity challenges:

  • Problem: Cross-reactivity with related endogenous retroviral proteins

  • Solution: Validate with multiple antibodies targeting different epitopes

  • Control: Include ERVFRD-1 knockdown samples as negative controls

• Background reduction strategies:

  • Problem: High background when using biotin-conjugated antibodies

  • Solution: Block endogenous biotin using avidin/biotin blocking kits

  • Alternative: Consider non-biotin detection systems if background persists

• Quantification challenges:

  • Problem: Variable expression levels across tissue samples

  • Solution: Use digital image analysis software for objective quantification

  • Standardization: Include calibration controls in each experiment

How can researchers optimize ERVFRD-1 immunoprecipitation protocols?

For researchers performing co-immunoprecipitation with ERVFRD-1:

• Lysis buffer optimization:

  • Use mild detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions

  • Include protease and phosphatase inhibitors to prevent degradation

  • Adjust salt concentration (150-300mM NaCl) based on interaction strength

• Antibody selection and coupling:

  • Test multiple antibodies targeting different epitopes

  • Use direct coupling to beads for cleaner results

  • Consider crosslinking antibodies to beads to prevent antibody leaching

• Washing optimization:

  • Balance stringency to remove non-specific binding while preserving specific interactions

  • Use graduated washing with increasing salt concentrations

  • Test different detergent concentrations in wash buffers

• Elution strategies:

  • For biotin-conjugated antibodies, use competitive elution with excess biotin

  • Alternative: use gentle elution with mild acids or basic solutions

  • For detecting binding partners, consider on-bead digestion for mass spectrometry

What emerging technologies might enhance ERVFRD-1 research?

Several cutting-edge technologies hold promise for advancing ERVFRD-1 research:

• Single-cell analysis applications:

  • Single-cell RNA sequencing to characterize heterogeneous expression patterns

  • Single-cell ATAC-seq to assess chromatin accessibility at the ERVFRD-1 locus

  • Spatial transcriptomics to map expression patterns within complex tissues

• CRISPR-based functional screening:

  • CRISPR activation/inhibition of ERVFRD-1 for functional studies

  • CRISPR tiling screens to identify regulatory elements

  • Base editing to study specific variants (e.g., rs9393931) without altering gene context

• Advanced imaging techniques:

  • Live-cell imaging with fluorescently tagged ERVFRD-1 to track trafficking

  • Super-resolution microscopy for subcellular localization

  • Correlative light and electron microscopy to study ultrastructural features

• Computational approaches:

  • Machine learning algorithms to identify expression patterns across cancer datasets

  • Network analysis to identify ERVFRD-1 functional partners

  • Integrative multi-omics analysis incorporating genomic, transcriptomic, and proteomic data

How might ERVFRD-1 research impact understanding of evolutionary biology?

ERVFRD-1 research offers unique insights into evolutionary biology:

• Comparative genomic approaches:

  • Analysis of ERVFRD-1 orthologs across species (reported in mouse and chimpanzee)

  • Evolutionary rate analysis to identify selection pressures

  • Investigation of co-evolution with interaction partners

• Functional conservation studies:

  • Cross-species comparison of syncytialization function

  • Analysis of immune modulatory functions across evolutionary lineages

  • Assessment of tissue-specific expression patterns in different species

• Evolutionary domestication research:

  • Investigation of retroviral gene co-option mechanisms

  • Comparative analysis with independently acquired syncytins in other mammalian lineages

  • Study of convergent evolution in placental development

• Methodological approaches:

  • Ancestral sequence reconstruction to infer evolutionary trajectories

  • Experimental testing of ancestral protein functions

  • Molecular dating techniques to establish timeline of ERVFRD-1 domestication