ERVFRD-1 Antibody

What is ERVFRD-1 and what are its key structural characteristics?

ERVFRD-1 (endogenous retrovirus group FRD member 1, envelope) is a human protein with 538 amino acid residues and a molecular mass of approximately 59.5 kDa. It is primarily localized in the cell membrane and belongs to the Gamma type-C retroviral envelope protein family. The protein undergoes several post-translational modifications, including protein cleavage and glycosylation, which can affect antibody recognition and experimental outcomes. ERVFRD-1 is also known by several synonyms including HERV-FRD provirus ancestral Env polyprotein, HERV-FRD_6p24.1 provirus ancestral Env polyprotein, syncytin-2, and syncytin 2 . Understanding these characteristics is crucial when designing experiments that utilize ERVFRD-1 antibodies.

What is the tissue distribution pattern of ERVFRD-1 expression?

ERVFRD-1 demonstrates a distinct tissue-specific expression pattern, with notably high expression in placental tissue. This differential expression is important to consider when selecting positive control tissues for antibody validation experiments. Recent research has also reported altered expression patterns in certain pathological conditions, with generally lower expression observed in kidney renal clear cell carcinoma (KIRC) tumors compared to normal tissue . When planning experiments with ERVFRD-1 antibodies, researchers should be aware of these expression patterns to properly interpret their results and choose appropriate positive and negative control tissues.

What biological functions has ERVFRD-1 been associated with?

ERVFRD-1 has been implicated in several important biological processes. In placental development, it contributes to the formation of the syncytiotrophoblast through cell-cell fusion events. Additionally, research suggests ERVFRD-1 plays a significant role in immunoregulation, particularly in tumor microenvironments. Studies have identified correlations between ERVFRD-1 expression and the infiltration levels of specific immune cells, including mast cells and regulatory T cells (Tregs) in KIRC . These findings suggest ERVFRD-1 may function as an immunomodulator in both physiological and pathological contexts, making it an interesting target for immunological research.

What are the primary research applications for ERVFRD-1 antibodies?

ERVFRD-1 antibodies find application across multiple experimental techniques, with Western blot being the most common application. Additional applications include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and immunofluorescence (IF) techniques . The table below summarizes the major applications and their specific considerations:

How should researchers optimize Western blot protocols for ERVFRD-1 detection?

When optimizing Western blot protocols for ERVFRD-1 detection, several factors require careful consideration. First, since ERVFRD-1 is a membrane protein, effective extraction requires specialized lysis buffers containing appropriate detergents. Second, the glycosylation state of ERVFRD-1 can affect its apparent molecular weight and potentially mask epitopes recognized by certain antibodies. Third, optimization of transfer conditions may be necessary for efficient transfer of this membrane protein. For primary antibody incubation, starting with a 1:500 dilution and adjusting based on signal-to-noise ratio is recommended. Secondary antibody selection should match the host species of the primary antibody, typically rabbit for most commercial ERVFRD-1 antibodies . Importantly, both positive controls (placental tissue extracts) and negative controls should be included to validate specificity.

What strategies can resolve weak or inconsistent ERVFRD-1 antibody signals?

Weak or inconsistent signals when using ERVFRD-1 antibodies may stem from several factors that require systematic troubleshooting. First, verify protein extraction efficiency using alternative extraction methods optimized for membrane proteins. Second, consider that glycosylation patterns may interfere with antibody binding; enzymatic deglycosylation with PNGase F before Western blotting may improve detection. Third, increase protein loading amounts or extend primary antibody incubation time (overnight at 4°C may yield better results than shorter incubations). Fourth, try signal enhancement systems such as biotin-streptavidin amplification or more sensitive chemiluminescent substrates. Finally, consider testing alternative antibodies that target different epitopes of ERVFRD-1, as epitope accessibility may vary depending on protein conformation and experimental conditions .

How can researchers validate the specificity of ERVFRD-1 antibodies?

Validating antibody specificity is crucial for generating reliable research data. For ERVFRD-1 antibodies, a multi-tiered validation approach is recommended. First, perform side-by-side testing of multiple antibodies targeting different epitopes of ERVFRD-1 to compare detection patterns. Second, include genetic validation through siRNA knockdown or CRISPR knockout of ERVFRD-1 to confirm signal reduction or elimination. Third, use recombinant ERVFRD-1 protein as a competitive inhibitor in immunostaining experiments to demonstrate binding specificity. Fourth, perform peptide blocking experiments using the immunizing peptide when available. Fifth, consider cross-reactivity testing against other members of the endogenous retrovirus family, particularly those with sequence similarity to ERVFRD-1. This comprehensive validation strategy helps ensure experimental observations truly reflect ERVFRD-1 biology rather than non-specific interactions .

What methodological approaches can reveal the relationship between ERVFRD-1 expression and immune cell infiltration in tumors?

Investigating ERVFRD-1's relationship with immune cell infiltration requires sophisticated methodological approaches. Multiplexed immunofluorescence or immunohistochemistry can visualize spatial relationships between ERVFRD-1-expressing cells and immune cell populations within the tumor microenvironment. Flow cytometry of disaggregated tumor samples allows quantitative assessment of multiple cell populations simultaneously. Computational approaches applied to transcriptomic data, such as single-sample Gene Set Enrichment Analysis (ssGSEA), can estimate immune cell infiltration scores and correlate these with ERVFRD-1 expression levels. Spearman's correlation analysis has been successfully employed to evaluate associations between ERVFRD-1 expression and various immune cell populations, revealing significant correlations with mast cells and regulatory T cells in KIRC . These multimodal approaches provide complementary insights into ERVFRD-1's immunoregulatory functions.

How can researchers investigate the methylation status of ERVFRD-1 and its impact on expression?

Epigenetic regulation, particularly DNA methylation, may play a crucial role in controlling ERVFRD-1 expression across different tissue types and disease states. Several methodological approaches can be employed to investigate this relationship. Bisulfite sequencing provides base-resolution methylation data for the ERVFRD-1 promoter region. Methylation-specific PCR offers a targeted approach for analyzing specific CpG sites. Genome-wide methylation arrays can place ERVFRD-1 methylation in broader epigenetic context. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) for methyl-binding proteins or histone modifications can reveal functional consequences of methylation. Statistical methods for correlation analysis between methylation status and expression levels include Pearson or Spearman correlation coefficients, with appropriate significance testing. Integration of methylation data with expression data is crucial for establishing regulatory relationships .

How might ERVFRD-1's immunoregulatory functions inform cancer immunotherapy research?

ERVFRD-1's demonstrated role in immunoregulation, particularly its relationship with mast cells and regulatory T cells, suggests potential applications in cancer immunotherapy research. Experimental approaches to explore this connection include in vitro co-culture systems with immune cells and ERVFRD-1-expressing tumor cells, with flow cytometry readouts of immune activation markers. In vivo models could assess whether modulation of ERVFRD-1 expression affects tumor immune infiltration or response to immune checkpoint inhibitors. Mechanistically, investigating whether ERVFRD-1 directly interacts with immune cell receptors or influences cytokine production would provide insights into its mode of action. These investigations may reveal whether ERVFRD-1 expression levels could predict immunotherapy response or whether targeting ERVFRD-1 directly might enhance anti-tumor immunity .

What techniques can elucidate the transcriptional regulation of ERVFRD-1 in different cellular contexts?

Understanding the transcriptional regulation of ERVFRD-1 requires sophisticated molecular techniques. Promoter analysis using luciferase reporter assays can identify key regulatory regions. ChIP-seq experiments targeting various transcription factors can reveal protein-DNA interactions at the ERVFRD-1 locus. CRISPR interference or activation systems can be employed to modulate specific regulatory elements and assess their impact on ERVFRD-1 expression. DNase I hypersensitivity assays or ATAC-seq can map chromatin accessibility at the ERVFRD-1 locus across different cell types. Integration of these data with transcription factor binding motif analysis can predict key regulatory proteins. Single-cell approaches may reveal cell-type specific regulatory mechanisms, particularly important in heterogeneous tissues or tumor samples where ERVFRD-1 expression varies across cell populations.

How can researchers address cross-reactivity concerns with ERVFRD-1 antibodies?

Cross-reactivity is a significant concern with antibodies targeting retroviral envelope proteins due to sequence similarities. When selecting an ERVFRD-1 antibody, researchers should review cross-reactivity testing data from manufacturers and conduct their own validation studies. Epitope mapping can identify antibodies targeting unique regions of ERVFRD-1 with minimal homology to related proteins. Pre-absorption experiments using recombinant related proteins can assess potential cross-reactivity. Mass spectrometry-based validation can provide definitive identification of immunoprecipitated proteins. When critical specificity is required, using multiple antibodies targeting different epitopes and comparing results can provide greater confidence in observations .

What strategies optimize immunohistochemical detection of ERVFRD-1 in tissue samples?

Optimizing immunohistochemical detection of ERVFRD-1 requires attention to several technical factors. Proper fixation is crucial; formalin fixation times should be standardized, as overfixation can mask epitopes. Antigen retrieval methods should be systematically compared, with both heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) and EDTA buffer (pH 9.0) as starting points. Blocking endogenous peroxidase activity is essential before applying primary antibody. Primary antibody concentration should be titrated, typically starting at 1:100 dilution and adjusting based on signal-to-noise ratio. Detection systems with signal amplification capabilities, such as polymer-based systems, may enhance sensitivity. Counterstaining should be optimized to provide context without obscuring specific signals. Automated staining platforms can improve reproducibility when available .