F2RL2 Antibody

What is F2RL2 and why is it significant in research?

F2RL2 (Coagulation factor II receptor-like 2) is a member of the G-protein coupled receptor 1 family that functions as a proteinase-activated receptor. It plays critical roles in coagulation pathways and cellular signaling processes. Research significance stems from its involvement in several pathological conditions, including myocardial infarction where F2RL2 downregulation has been shown to prevent reduction in ejection fraction and fractional shortening while reducing infarct area and cell apoptosis in mouse models . F2RL2 belongs to a superfamily of receptors that transmit signals across cell membranes in response to proteolytic cleavage of their extracellular domains, making them important targets for understanding disease mechanisms and potential therapeutic interventions .

What are the recommended applications for F2RL2 antibodies?

F2RL2 antibodies have been validated for several experimental applications with specific recommended dilutions:

These applications enable researchers to investigate F2RL2 expression patterns, localization, and quantification in various experimental models . The optimal working concentration should be determined by the researcher through titration experiments as antibody performance can vary depending on sample type and preparation methods.

How should F2RL2 antibodies be stored and handled for optimal results?

For optimal performance, F2RL2 antibodies should be stored at -20°C for long-term preservation (up to one year). For frequent use and short-term storage (up to one month), keeping the antibody at 4°C is recommended. It's crucial to avoid repeated freeze-thaw cycles as they can compromise antibody integrity and binding capacity . The typical formulation includes PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide, which helps maintain stability during storage . When working with the antibody, it's advisable to aliquot the stock solution to minimize freeze-thaw cycles and maintain consistent experimental conditions across studies.

How can F2RL2 antibodies be utilized in myocardial infarction research models?

Recent studies have demonstrated that F2RL2 plays a significant role in myocardial infarction (MI) pathophysiology. When designing MI research with F2RL2 antibodies, researchers should consider:

• Animal model validation: F2RL2 downregulation in MI mouse models has shown prevention of reduced ejection fraction and fractional shortening, indicating cardioprotective effects .

• Cell-based experimental design: Human cardiac myocytes (HCMs) subjected to oxygen-glucose deprivation (OGD) exhibit increased F2RL2 expression, which correlates with decreased cell viability and increased apoptosis. F2RL2 knockdown experiments reversed these effects .

• Regulatory pathway analysis: F2RL2 appears to be regulated by a lncRNA NEAT1/miR-582-5p axis, where NEAT1 sponges miR-582-5p, which normally targets F2RL2 . This suggests an experimental approach combining:

  • Immunofluorescence staining with F2RL2 antibodies

  • Western blot quantification of F2RL2 protein

  • qRT-PCR analysis of related gene expression

When investigating therapeutic interventions, researchers should monitor markers of cardiac function (EF and FS via echocardiography), infarct area (TTC and Evans blue staining), and apoptosis (TUNEL assay) in relation to F2RL2 expression levels .

What approaches should be considered when investigating F2RL2 in cancer research?

While F2RL2-specific cancer research data is limited in the provided search results, related research on F2R (Coagulation Factor II Receptor) offers valuable insights that may guide F2RL2 cancer investigations:

• Expression analysis strategy: Analyzing F2RL2 expression differences between tumor and normal tissues using public databases (TCGA, GEO) can identify potential biomarker value. Similar to F2R in gastric adenocarcinoma, researchers should correlate expression with clinical parameters and survival outcomes .

• Mechanistic investigation design: Consider the following experimental approaches:

  • Knockdown/overexpression studies with F2RL2 antibody validation

  • Cell proliferation, migration, and invasion assays

  • Correlation with stromal and immune cell infiltration

  • Pathway enrichment analysis (GO, KEGG, GSEA)

• Drug response correlation: Investigate potential correlations between F2RL2 expression and drug sensitivity, similar to F2R correlations with compounds like BEZ235, Dasatinib, and Rapamycin .

When designing such experiments, F2RL2 antibody validation through immunofluorescence or Western blotting is crucial for ensuring target specificity, particularly when evaluating expression changes in response to experimental manipulations.

How can researchers evaluate cross-reactivity of F2RL2 antibodies across different species?

Cross-reactivity evaluation is essential for comparative studies and translational research. Based on customer inquiries and technical support responses, the following methodological approach is recommended:

• Sequence homology analysis: Perform BLAST analysis comparing the immunogen sequence (aa 38-87 of human F2RL2) with the target species sequence to predict potential cross-reactivity .

• Pilot testing methodology: Even with high sequence homology, empirical validation is necessary. Design a small-scale pilot test incorporating:

  • Positive controls (human samples with known F2RL2 expression)

  • Samples from target species (e.g., goat, bovine)

  • Negative controls (tissues known to lack F2RL2 expression)

• Validation experiments: Use multiple techniques for comprehensive cross-reactivity assessment:

  • Western blot to confirm correct molecular weight detection

  • Immunofluorescence to verify localization patterns

  • Blocking peptide controls to confirm specificity

Customer inquiries about cross-reactivity with goat and bovine tissues indicate research interest in F2RL2 across diverse species, though validation data for these species was not established in the provided information .

What controls should be included when using F2RL2 antibodies in immunofluorescence studies?

Rigorous control implementation is critical for generating reliable immunofluorescence data with F2RL2 antibodies. Based on validation practices, researchers should include:

• Positive tissue/cell controls: MCF7 cells have been validated for positive F2RL2 expression and serve as effective positive controls in immunofluorescence experiments .

• Peptide blocking controls: Synthesized peptide blocking experiments are essential to confirm antibody specificity. The immunofluorescence analysis shown in the validation data demonstrates marked signal reduction when the F2RL2 antibody is pre-incubated with the synthesized peptide immunogen .

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Tissues/cells known to lack F2RL2 expression

• Expression validation controls: When investigating a new tissue type (such as retina, which was queried in the search results), researchers should first confirm F2RL2 expression through qRT-PCR or publicly available expression databases prior to antibody studies .

The dilution range of 1:200 to 1:1000 for immunofluorescence provides a starting point, but optimization for each specific application and tissue type is strongly recommended .

What considerations are important when using F2RL2 antibodies for Western blot analysis?

While Western blot protocols specific to F2RL2 were limited in the search results, the following methodological considerations are important based on the available information:

• Molecular weight expectations: The observed molecular weight of F2RL2 is approximately 72 kDa, which differs significantly from the calculated molecular weight of 42.5 kDa . This discrepancy is likely due to post-translational modifications and should be considered when interpreting bands.

• Protein extraction method: For optimal F2RL2 detection, RIPA lysis buffer extraction followed by BCA protein quantification at 562 nm has been successfully employed in research studies .

• Loading controls selection: When studying F2RL2 in disease models, appropriate loading controls should be selected based on the experimental context. For myocardial infarction studies, housekeeping proteins that remain stable during cardiac stress should be prioritized.

• Sample preparation considerations:

  • Complete denaturation is important for membrane proteins like F2RL2

  • Avoid excessive heating which may cause protein aggregation

  • Consider using fresh samples when possible, as F2RL2 stability during long-term storage may vary

• Validation approach: When using F2RL2 antibodies in new experimental contexts (e.g., retina research mentioned in customer inquiries), method validation should include positive controls and correlation with known expression patterns .

How should researchers optimize F2RL2 antibody dilutions for different experimental applications?

Optimization of antibody dilutions is critical for achieving the ideal signal-to-noise ratio while conserving reagents. Based on the manufacturer's recommendations and research applications, the following methodological approach is suggested:

• Starting dilution guidelines:

  • ICC/IF: Begin with 1:500 (middle of the 1:200-1:1000 recommended range)

  • ELISA: Start with 1:10,000 as recommended

  • Western blot: Although specific recommendations weren't provided, begin with 1:1000 based on typical antibody applications

• Titration experimental design:

  • Prepare a dilution series (e.g., 1:100, 1:500, 1:1000, 1:5000)

  • Use identical samples across all dilutions

  • Maintain consistent experimental conditions (incubation time, temperature, detection method)

  • Include appropriate positive and negative controls

• Evaluation criteria:

  • Signal specificity (absence of signal in negative controls)

  • Signal intensity (sufficient for reliable detection)

  • Background levels (minimal non-specific staining)

  • Signal-to-noise ratio (quantitative assessment)

• Application-specific considerations:

  • For ELISA: Consider coating concentration and blocking efficiency

  • For IF: Background autofluorescence of specific tissues must be accounted for

  • For ICC: Cell fixation method may influence optimal antibody concentration

Optimal dilutions may vary between different lots of the same antibody, so verification is recommended when switching to a new lot .

How can researchers address cross-reactivity issues when working with F2RL2 antibodies in novel tissue types?

When applying F2RL2 antibodies to novel tissue types such as retina or bovine thymus (as mentioned in customer inquiries), researchers may encounter cross-reactivity challenges. The following methodological approach can help address these issues:

• Pre-experimental assessment:

  • Analyze F2RL2 expression in the target tissue using transcriptomic databases

  • Perform sequence alignment between the immunogen region (aa 38-87) and potential cross-reactive proteins

  • Review literature for reported F2RL2 expression patterns in related tissues

• Experimental validation strategy:

  • Multiple detection methods: Compare results from IF/ICC with Western blot or qRT-PCR

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm signal specificity

  • Antibody concentration gradient: Test multiple dilutions to identify optimal signal-to-noise ratio

  • Inclusion of known positive and negative control tissues

• Advanced specificity controls:

  • siRNA or CRISPR-based F2RL2 knockdown in cell models

  • F2RL2 overexpression systems for positive control generation

  • Use of tissue from F2RL2 knockout animals (if available)

When investigating F2RL2 in retina, researchers should note that F2RL2 appears to be highly expressed in retinal tissue, suggesting that antibody validation in this context may yield positive results but requires careful specificity controls .

What factors might explain discrepancies between observed and calculated molecular weights for F2RL2?

The significant difference between the observed molecular weight of F2RL2 (72 kDa) and its calculated weight (42.5 kDa) noted in the product information warrants careful consideration when interpreting Western blot results. Researchers should consider the following explanations and verification approaches:

• Post-translational modifications:

  • Glycosylation: G-protein coupled receptors like F2RL2 often undergo extensive glycosylation

  • Phosphorylation: Multiple phosphorylation sites may alter migration

  • Ubiquitination: Potential ubiquitination may significantly increase apparent molecular weight

• Technical verification approaches:

  • Enzymatic deglycosylation: Treatment with PNGase F or similar enzymes to remove N-linked glycans

  • Phosphatase treatment: To remove phosphate groups

  • Sample preparation variations: Different detergents or reducing conditions to rule out aggregation

• Alternative splicing consideration:

  • Analyze database entries for known F2RL2 splice variants

  • Consider primers that can detect specific splice variants in companion qRT-PCR experiments

• Experimental controls:

  • Recombinant F2RL2 protein as size reference

  • Side-by-side comparison with other validated F2RL2 antibodies

  • Immunoprecipitation followed by mass spectrometry for definitive identification

Understanding these factors is crucial for accurate data interpretation, especially when working with F2RL2 in novel experimental contexts or when comparing results across different studies.

How should researchers integrate F2RL2 antibody data with gene expression analysis in complex pathway studies?

When investigating F2RL2 in regulatory pathways, such as the NEAT1/miR-582-5p/F2RL2 axis in myocardial infarction or potential roles in cancer , integration of protein and gene expression data requires methodological rigor:

• Multi-level experimental design:

  • Protein level: Western blot and immunostaining with F2RL2 antibodies

  • mRNA level: qRT-PCR for F2RL2 transcript quantification

  • Regulatory level: Analysis of upstream regulators (e.g., miR-582-5p, NEAT1)

  • Functional level: Phenotypic assays related to the pathway under investigation

• Correlation analysis methodology:

  • Pearson or Spearman correlation between F2RL2 protein levels and mRNA expression

  • Temporal analysis of expression changes following pathway perturbation

  • Spatial correlation in tissue sections using co-localization studies

• Pathway validation approaches:

  • Sequential manipulation experiments (e.g., NEAT1 silencing → measure miR-582-5p → measure F2RL2)

  • Rescue experiments to confirm pathway directionality

  • Reporter assays to confirm direct regulatory relationships

• Data integration framework:

  • Use consistent normalization methods across experiments

  • Apply appropriate statistical models for multi-omics data integration

  • Consider using pathway analysis tools (GSEA, IPA, etc.) for contextualizing findings

In the specific context of myocardial infarction research, researchers should correlate F2RL2 antibody data with functional cardiac parameters (EF, FS), infarct measurements, and molecular markers of apoptosis to comprehensively understand F2RL2's role in the disease mechanism .

What emerging applications of F2RL2 antibodies are being explored in translational research?

Based on the current research landscape, several promising translational applications of F2RL2 antibodies are emerging:

• Cardiovascular disease biomarkers: The demonstrated role of F2RL2 in myocardial infarction models suggests potential for developing F2RL2-based prognostic or diagnostic tools. Research could focus on correlating F2RL2 expression with disease severity and treatment response in patient samples.

• Cancer research applications: Given the findings on related receptor F2R in gastric adenocarcinoma , investigation of F2RL2 in various cancer types represents a logical extension. F2RL2 antibodies could enable tissue microarray studies across cancer types to identify potential diagnostic or therapeutic targets.

• Therapeutic target validation: The knockdown studies showing beneficial effects of F2RL2 downregulation in myocardial infarction suggest that F2RL2 may be a viable therapeutic target. Antibody-based validation of small molecule inhibitors or biologics targeting F2RL2 could advance drug development efforts.

• Receptor signaling mechanisms: Further investigation into the downstream signaling pathways of F2RL2 activation could reveal novel intervention points. Co-immunoprecipitation studies using F2RL2 antibodies could identify interaction partners and signaling complexes.