FREM1 Antibody

What is FREM1 and why is it significant as a research target?

FREM1 is an extracellular matrix protein involved in the formation and organization of basement membranes, which are thin sheet-like structures that separate and support cells in many tissues. The protein interacts with FRAS1 and FREM2 during embryonic development as components of basement membranes. These basement membranes anchor epithelial cells to other embryonic tissues, including those that develop into connective tissues and kidneys . FREM1 has gained significance as a research target due to its associations with multiple conditions, including its identification as a diagnostic gene signal in heart failure progression and its potential role in conferring resistance to HIV infection . Additionally, mutations in FREM1 have been linked to Manitoba oculotrichoanal syndrome and bifid nose, renal agenesis, and anorectal malformations syndrome .

What are the primary applications of FREM1 antibodies in research?

FREM1 antibodies are utilized in multiple research applications focusing on both basic science and translational medicine:

• Western Blotting (WB): Detection of FREM1 protein expression in tissue and cell lysates with recommended working dilutions of 0.5-1 μg/mL

• Immunohistochemistry (IHC-P): Visualization of FREM1 in paraffin-embedded tissue sections at approximately 10 μg/mL concentrations

• Immunocytochemistry (ICC): Detection of FREM1 in cultured cells

• ELISA: Quantitative measurement of FREM1 protein levels

FREM1 antibodies have been particularly valuable in cardiovascular research to investigate heart failure mechanisms and in HIV research to understand resistance mechanisms in mucosal tissues .

What tissue expression patterns should researchers expect when using FREM1 antibodies?

FREM1 shows a specific expression pattern that researchers should be aware of when planning experiments:

• Embryonic tissues: FREM1 is widely expressed in regions of epithelial/mesenchymal interaction and epidermal remodeling during development

• Adult tissues: FREM1 mRNA is highly expressed in tissues relevant for HIV-1 infection, with protein expression detected in the ectocervical mucosa of HIV-resistant women

• Cardiovascular system: Significant expression has been observed in heart tissue, where FREM1 may serve as a diagnostic gene signal for heart failure

• Immune context: FREM1 expression has been correlated with specific immune cell subtypes, showing positive correlation with resting mast cells (r = 0.353, P < 0.001) and negative correlation with neutrophils (r = −0.270, P < 0.001)

For immunohistochemical detection, researchers should anticipate signal in basement membrane regions where epithelial cells interface with underlying tissues.

How can FREM1 antibodies be used to investigate heart failure pathogenesis?

FREM1 antibodies can be employed in sophisticated research designs to investigate heart failure mechanisms. Recent integrated bioinformatics approaches have identified FREM1 as a diagnostic gene signal in heart failure progression with exceptional potential (AUC = 0.953-1.000 in validation datasets) .

Methodological approach:

• Tissue comparison analysis: Use FREM1 antibodies in immunohistochemistry to compare heart tissue from normal and heart failure subjects

• Correlation with immune infiltrates: Combine FREM1 immunostaining with immune cell markers to verify the significant correlations observed with:

  • Resting mast cells (positive correlation, r = 0.353, P < 0.001)

  • Neutrophils (negative correlation, r = −0.270, P < 0.001)

  • T cell subtypes and macrophage populations

The following table summarizes immune cell populations significantly altered in heart failure that correlate with FREM1 expression:

This approach allows researchers to investigate how FREM1 expression relates to immune cell infiltration and activation in heart failure pathogenesis .

What methods should be used to investigate FREM1's role in HIV resistance mechanisms?

To investigate FREM1's role in HIV resistance, researchers should employ a multi-faceted approach combining genetic, protein expression, and functional analyses. Genetic evidence has associated the minor allele of SNP rs1552896 in FREM1 with resistance to HIV infection (OR = 2.67, 95% CI: 1.47-4.84) .

Recommended methodological framework:

• Genetic screening: Genotype rs1552896 and other FREM1 polymorphisms in study populations with differential HIV susceptibility

• Protein localization: Perform immunohistochemistry using FREM1 antibodies on ectocervical mucosal biopsies, where FREM1 protein has been shown to be expressed in HIV-resistant women

• Expression quantification: Use Western blotting and qRT-PCR to quantify FREM1 protein and mRNA levels in relevant tissues

• Functional assays: Develop in vitro models of HIV entry and infection in cells with modulated FREM1 expression

Researchers should be aware that FREM1 has a splice variant called TILRR (Toll-like interleukin-1 receptor regulator) that is an integral component of innate immune responses . Both forms should be investigated, as TILRR can stimulate innate immune responses and its expression increases in monocytes and hardened plaques after myocardial infarction .

What are the optimal conditions for using FREM1 antibodies in immunohistochemistry of different tissue types?

Successful immunohistochemical detection of FREM1 requires careful optimization based on tissue type and fixation method:

For paraffin-embedded tissues:

• Antigen retrieval: Use citric acid-based antigen retrieval methods as demonstrated in successful studies of FREM1 expression

• Antibody concentration: Begin with 10 μg/mL for paraffin sections and adjust based on signal-to-noise ratio

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature

• Detection systems: For tissues with lower expression levels, amplification systems such as tyramide signal amplification may be necessary

• Controls: Include both positive controls (tissues known to express FREM1) and negative controls (either primary antibody omission or isotype controls)

For basement membrane visualization, dual immunofluorescence with antibodies against collagen or other basement membrane components can help localize FREM1 in relation to the basement membrane structure .

How can researchers troubleshoot non-specific binding when using FREM1 antibodies?

Non-specific binding is a common challenge when working with antibodies against extracellular matrix proteins like FREM1. Researchers can employ these strategies to improve specificity:

• Antibody selection: Choose affinity-purified antibodies, such as those raised against specific peptides near the carboxy terminus of human FREM1

• Blocking optimization: Extend blocking steps (2-3 hours at room temperature) using 5-10% normal serum from the species in which the secondary antibody was raised

• Buffer adjustment: Add 0.1-0.3% Triton X-100 for intracellular epitopes or reduce detergent concentration for membrane proteins

• Cross-adsorption: If cross-reactivity with related proteins (FRAS1, FREM2) is suspected, pre-adsorb the antibody with recombinant related proteins

• Validation approaches: Confirm specificity through multiple methods:

  • Testing in tissues from FREM1 knockout models

  • Peptide competition assays

  • Comparing staining patterns with multiple antibodies targeting different FREM1 epitopes

For Western blotting applications, increasing the washing duration and stringency can help reduce background signals.

How can FREM1 antibodies be used to investigate the relationship between FREM1 and immune cell function?

Emerging research has revealed significant correlations between FREM1 expression and immune cell populations, suggesting new research opportunities:

• Immune cell correlation studies: Use flow cytometry with FREM1 antibodies to analyze FREM1 expression in sorted immune cell populations, especially focusing on:

  • Mast cells (strongest positive correlation with FREM1)

  • Neutrophils (strongest negative correlation with FREM1)

  • T cell subpopulations

  • Macrophage subtypes

• Functional impact assessment: Investigate how FREM1 modulates immune cell function by:

  • Analyzing cytokine production in cells with differential FREM1 expression

  • Assessing migration and adhesion properties of immune cells in the presence of recombinant FREM1

  • Evaluating FREM1's role in immune cell recruitment to sites of inflammation

• TILRR variant studies: Specifically investigate the TILRR splice variant of FREM1, which has been shown to regulate immune inflammation and stimulate innate immune responses

The relationship between FREM1 and immune cells is particularly relevant for both cardiovascular disease and infectious disease research, as FREM1 appears to influence inflammatory responses in multiple contexts.

What is the potential for FREM1 as a diagnostic biomarker in cardiovascular disease?

Integrated bioinformatics approaches have identified FREM1 as a promising diagnostic gene signal for heart failure with exceptional performance metrics. Researchers can explore its biomarker potential using these approaches:

• Biomarker validation studies: Design studies to validate FREM1 as a diagnostic biomarker by:

  • Measuring FREM1 expression in large cohorts of heart failure patients versus controls

  • Correlating FREM1 levels with disease severity, progression, and outcomes

  • Comparing FREM1's diagnostic value to established biomarkers (BNP, NT-proBNP)

• Multi-omics integration: Combine antibody-based protein detection with:

  • Transcriptomic data (RNA-seq)

  • Genetic information (relevant SNPs)

  • Proteomic profiles

• Development of diagnostic assays: Create standardized ELISA or other immunoassays specifically optimized for FREM1 detection in clinical samples

Machine learning approaches have already shown the value of FREM1 as a diagnostic signal. In validation studies, FREM1 demonstrated remarkable diagnostic performance with AUC values of 0.953 (95% CI: 0.904-1.000) and 1.000 (95% CI: 1.000-1.000) in two independent datasets . This exceptional performance suggests that antibody-based detection of FREM1 could provide valuable clinical diagnostic information.